Pharmaceutical Composition Comprising Anti-Mirna Antisense Oligonucleotides

ABSTRACT

The invention provides pharmaceutical compositions comprising short single stranded oligonucleotides, of length of between 8 and 26 nucleobases which are complementary to human microRNAs selected from the group consisting of miR19b, miR21, miR122a, miR155 and miR375. The short oligonucleotides are particularly effective at alleviating miRNA repression in vivo. It is found that the incorporation of high affinity nucleotide analogues into the oligonucleotides results in highly effective anti-microRNA molecules which appear to function via the formation of almost irreversible duplexes with the miRNA target, rather than RNA cleavage based mechanisms, such as mechanisms associated with RNaseH or RISC.

FIELD OF THE INVENTION

The present invention concerns pharmaceutical compositions comprisingLNA-containing single stranded oligonucleotides capable of inhibitingdisease-inducing microRNAs particularly human micro-RNAs miR-19b,miR-21, miR-122A, miR-155 and miR-375.

BACKGROUND OF THE INVENTION

MicroRNAs—Novel Regulators of Gene Expression

MicroRNAs (miRNAs) are an abundant class of short endogenous RNAs thatact as post-transcriptional regulators of gene expression bybase-pairing with their target mRNAs. The mature miRNAs are processedsequentially from longer hairpin transcripts by the RNAse IIIribonucleases Drosha (Lee et al. 2003) and Dicer (Hutvagner et al. 2001,Ketting et al. 2001). To date more than 3400 miRNAs have been annotatedin vertebrates, invertebrates and plants according to the miRBasemicroRNA database release 7.1 in October 2005 (Griffith-Jones 2004,Griffith-Jones et al. 2006), and many miRNAs that correspond to putativegenes have also been identified.

Most animal miRNAs recognize their target sites located in 3′-UTRs byincomplete base-pairing, resulting in translational repression of thetarget genes (Bartel 2004). An increasing body of research shows thatanimal miRNAs play fundamental biological roles in cell growth andapoptosis (Brennecke et al. 2003), hematopoietic lineage differentiation(Chen et al. 2004), life-span regulation (Boehm and Slack 2005),photoreceptor differentiation (Li and Carthew 2005), homeobox generegulation (Yekta et al. 2004, Hornstein et al. 2005), neuronalasymmetry (Johnston et al. 2004), insulin secretion (Poy et al. 2004),brain morphogenesis (Giraldez et al. 2005), muscle proliferation anddifferentiation (Chen, Mandel et al. 2005, Kwon et al. 2005, Sokol andAmbros 2005), cardiogenesis (Zhao et al. 2005) and late embryonicdevelopment in vertebrates (Wienholds et al. 2005).

MicroRNAs in Human Diseases

miRNAs are involved in a wide variety of human diseases. One is spinalmuscular atrophy (SMA), a paediatric neurodegenerative disease caused byreduced protein levels or loss-of-function mutations of the survival ofmotor neurons (SMN) gene (Paushkin et al. 2002). A mutation in thetarget site of miR-189 in the human SLITRK1 gene was recently shown tobe associated with Tourette's syndrome (Abelson et al. 2005), whileanother recent study reported that the hepatitis C virus (HCV) RNAgenome interacts with a host-cell microRNA, the liver-specific miR-122a,to facilitate its replication in the host (Jopling et al. 2005). Otherdiseases in which miRNAs or their processing machinery have beenimplicated, include fragile X mental retardation (FXMR) caused byabsence of the fragile X mental retardation protein (FMRP) (Nelson etal. 2003, Jin et al. 2004) and DiGeorge syndrome (Landthaler et al.2004).

In addition, perturbed miRNA expression patterns have been reported inmany human cancers. For example, the human miRNA genes miR15a andmiR16-1 are deleted or down-regulated in the majority of B-cell chroniclymphocytic leukemia (CLL) cases, where a unique signature of 13 miRNAgenes was recently shown to associate with prognosis and progression(Calin et al. 2002, Calin et al. 2005). The role of miRNAs in cancer isfurther supported by the fact that more than 50% of the human miRNAgenes are located in cancer-associated genomic regions or at fragilesites (Calin et al. 2004). Recently, systematic expression analysis of adiversity of human cancers revealed a general down-regulation of miRNAsin tumors compared to normal tissues (Lu et al. 2005). Interestingly,miRNA-based classification of poorly differentiated tumors wassuccessful, whereas mRNA profiles were highly inaccurate when applied tothe same samples. miRNAs have also been shown to be deregulated inbreast cancer (Iorio et al. 2005), lung cancer (Johnson et al. 2005) andcolon cancer (Michael et al. 2004), while the miR-17-92 cluster, whichis amplified in human B-cell lymphomas and miR-155 which is upregulatedin Burkitt's lymphoma have been reported as the first human miRNAoncogenes (Eis et al. 2005, He et al. 2005). Thus, human miRNAs wouldnot only be highly useful as biomarkers for future cancer diagnostics,but are rapidly emerging as attractive targets for disease interventionby oligonucleotide technologies.

Inhibition of microRNAs Using Single Stranded Oligonucleotides

Several oligonucleotide approaches have been reported for inhibition ofmiRNAs.

WO03/029459 (Tuschl) claims oligonucleotides which encode microRNAs andtheir complements of between 18-25 nucleotides in length which maycomprise nucleotide analogues. LNA is suggested as a possible nucleotideanalogue, although no LNA containing olginucleotides are disclosed.Tuschl claims that miRNA oligonucleotides may be used in therapy.

US2005/0182005 discloses a 24 mer 2′OMe RNA oligoribonucleotidecomplementary to the longest form of miR 21 which was found to reducemiR 21 induced repression, whereas an equivalent DNA containingoligonucleotide did not. The term 2′OMe-RNA refers to an RNA analoguewhere there is a substitution to methyl at the 2′ position (2′OMethyl).

US2005/0227934 (Tuschl) refers to antimir molecules with up to 50% DNAresidues. It also reports that antimirs containing 2′OMe RNA were usedagainst pancreatic microRNAs but it appears that no actualoligonucleotide structures are disclosed.

US20050261218 (ISIS) claims an oligomeric compound comprising a firstregion and a second region, wherein at least one region comprises amodification and a portion of the oligomeric compound is targeted to asmall non-coding RNA target nucleic acid, wherein the small non-codingRNA target nucleic acid is a miRNA. Oligomeric compounds of between 17and 25 nucleotides in length are claimed. The examples refer to entirely2′OMe PS compounds, 21 mers and 20 mers, and 2′OMe gapmeroligonucleotides targeted against a range of pre-miRNA and mature miRNAtargets.

Boutla et al. 2003 (Nucleic Acids Research 31: 4973-4980) describe theuse of DNA antisense oligonucleotides complementary to 11 differentmiRNAs in Drosophila as well as their use to inactivate the miRNAs byinjecting the DNA oligonucleotides into fly embryos. Of the 11 DNAantisense oligonucleotides, only 4 constructs showed severe interferencewith normal development, while the remaining 7 oligonucleotides didn'tshow any phenotypes presumably due to their inability to inhibit themiRNA in question.

An alternative approach to this has been reported by Hutvagner et al.(2004) and Leaman et al. (2005), in which 2′-O-methyl antisenseoligonucleotides, complementary to the mature miRNA could be used aspotent and irreversible inhibitors of short interfering RNA (siRNA) andmiRNA function in vitro and in vivo in Drosophila and C. elegans,thereby inducing a loss-of-function phenotype. A drawback of this methodis the need of high 2′-O-methyl oligonucleotide concentrations (100micromolar) in transfection and injection experiments, which may betoxic to the animal. This method was recently applied to mice studies,by conjugating 2′-O-methyl antisense oligonucleotides complementary tofour different miRNAs with cholesterol for silencing miRNAs in vivo(Krützfedt et al. 2005). These so-called antagomirs were administered tomice by intravenous injections. Although these experiments resulted ineffective silencing of endogenous miRNAs in vivo, which was found to bespecific, efficient and long-lasting, a major drawback was the need ofhigh dosage (80 mg/kg) of 2′-O-Me antagomir for efficient silencing.

Inhibition of microRNAs using LNA-modified oligonucleotides havepreviously been described by Chan et al. Cancer Research 2005, 65 (14)6029-6033, Lecellier et al. Science 2005, 308, 557-560, Naguibneva etal. Nature Cell Biology 2006 8 (3), 278-84 and Ørum et al. Gene 2006,(Available online 24 Feb. 2006). In all cases, the LNA-modified anti-miroligonucleotides were complementary to the entire mature microRNA, i.e.20-23 nucleotides in length, which hampers efficient in vivo uptake andwide biodistribution of the molecules.

Naguibneva (Naguibneva et al. Nature Cell Biology 2006 8 describes theuse of mixmer DNA-LNA-DNA antisense oligonucleotide anti-mir to inhibitmicroRNA miR-181 function in vitro, in which a block of 8 LNAnucleotides is located at the center of the molecule flanked by 6 DNAnucleotides at the 5′ end, and 9 DNA nucleotides at the 3′ end,respectively. A major drawback of this antisense design is low in vivostability due to low nuclease resistance of the flanking DNA ends.

While Chan et al. (Chan et al. Cancer Research 2005, 65 (14) 6029-6033),and Ørum et al. (Ørum et al. Gene 2006, (Available online 24 Feb. 2006)do not disclose the design of the LNA-modified anti-mir molecules usedin their study, Lecellier et al. (Lecellier et al. Science 2005, 308,557-560) describes the use of gapmer LNA-DNA-LNA antisenseoligonucleotide anti-mir to inhibit microRNA function, in which a blockof 4 LNA nucleotides is located both at the 5′ end, and at the 3′ end,respectively, with a window of 13 DNA nucleotides at the center of themolecule. A major drawback of this antisense design is low in vivouptake, as well as low in vivo stability due to the 13 nucleotide DNAstretch in the anti-mir oligonucleotide.

Thus, there is a need in the field for improved oligonucleotides capableof inhibiting microRNAs.

SUMMARY OF THE INVENTION

The present invention is based upon the discovery that the use of shortoligonucleotides designed to bind with high affinity to miRNA targetsare highly effective in alleviating the repression of mRNA by microRNAsin vivo.

Whilst not wishing to be bound to any specific theory, the evidencedisclosed herein indicates that the highly efficient targeting of miRNAsin vivo is achieved by designing oligonucleotides with the aim offorming a highly stable duplex with the miRNA target in vivo. This isachieved by the use of high affinity nucleotide analogues such as atleast one LNA units and suitably further high affinity nucleotideanalogues, such as LNA, 2′-MOE RNA of 2′-Fluoro nucleotide analogues, ina short, such as 10-17 or 10-16 nucleobase oligonucleotides. In oneaspect the aim is to generate an oligonucleotide of a length which isunlikely to form a siRNA complex (i.e. a short oligonucleotide), andwith sufficient loading of high affinity nucleotide analogues that theoligonucleotide sticks almost permanently to its miRNA target,effectively forming a stable and non-functional duplex with the miRNAtarget. We have found that such designs are considerably more effectivethan the prior art oligonucleotides, particularly gapmer and blockmerdesigns and oligonucleotides which have a long length, e.g. 20-23 mers.The term 2′fluor-DNA refers to a DNA analogue where the is asubstitution to fluor at the 2′ position (2′F).

The invention provides a pharmaceutical composition comprising anoligonucleotide having a length of between 8 and 17, such as 10 and 17,such as 8-16 or 10-16 nucleobase units, a pharmaceutically acceptablediluent, carrier, or adjuvant, wherein at least one of the nucleobaseunits of the single stranded oligonucleotide is a high affinitynucleotide analogue, such as a Locked Nucleic Acid (LNA) nucleobaseunit, and wherein the single stranded oligonucleotide is complementaryto a human microRNA sequence selected from the group consisting of humanmicro-RNAs miR-19b, miR-21, miR-122A, miR-155 and miR-375.

The invention provides for a pharmaceutical composition comprising anoligonucleotide having a length of from 10 to 26 nucleobase units, and apharmaceutically acceptable diluent, carrier, or adjuvant, wherein theoligonucleotide comprises a core DNA sequence from positions two toseven or from positions three to eight, counting from the 3′ end of 3′acgttt 5′ (SEQ ID NO 6, 5′tttgca3′), wherein at least one, such as one,preferably at least two, such as two or three, DNA units in saidsequence have been substituted by their corresponding LNA unit andoptionally wherein said oligonucleotide does not comprise a region ofmore than 7 contiguous DNA units.

The invention provides for a pharmaceutical composition comprising anoligonucleotide having a length of from 10 to 26 nucleobase units, and apharmaceutically acceptable diluent, carrier, or adjuvant, wherein theoligonucleotide comprises a core DNA sequence from positions two toseven or from positions three to eight, counting from the 3′ end of 3′ctcaca 5′ (SEQ ID NO 7, 5′ acactc 3′) wherein at least one, such as one,preferably at least two, such as two or three, DNA units in saidsequence have been substituted by their corresponding LNA unit andoptionally wherein said oligonucleotide does not comprise a region ofmore than 7 contiguous DNA units.

The invention provides for a pharmaceutical composition comprising anoligonucleotide having a length of from 10 to 26 nucleobase units, and apharmaceutically acceptable diluent, carrier, or adjuvant, wherein theoligonucleotide comprises a core DNA sequence from positions two toseven or from positions three to eight, counting from the 3′ end of

3′ ttacga 5′ (SEQ ID NO 8, 5′agcatt3′) wherein at least one, such asone, preferably at least two, such as two or three, DNA units in saidsequence have been substituted by their corresponding LNA unit andoptionally wherein said oligonucleotide does not comprise a region ofmore than 7 contiguous DNA units.

The invention provides for a pharmaceutical composition comprising asingle stranded oligonucleotide having a length of from 10 to 26nucleobase units, and a pharmaceutically acceptable diluent, carrier, oradjuvant, wherein the oligonucleotide comprises a core DNA sequence frompositions two to seven or from positions three to eight, counting fromthe 3′ end of 3′ acaagc 5′ (SEQ ID NO 9, 5′ cgaaca 3′) wherein at leastone, such as one, preferably at least two, such as two or three, DNAunits in said sequence have been substituted by their corresponding LNAunit and optionally wherein said oligonucleotide does not comprise aregion of more than 7 contiguous DNA units.

The invention provides for a pharmaceutical composition comprising asingle stranded oligonucleotide having a length of from 10 to 26nucleobase units, and a pharmaceutically acceptable diluent, carrier, oradjuvant, wherein the oligonucleotide comprises a core DNA sequence frompositions two to seven or from positions three to eight, counting fromthe 3′ end of 3′ cgaata 5′ (SEQ ID NO 10, 5′ ataagc3′) wherein at leastone, such as one, preferably at least two, such as two or three, DNAunits in said sequence have been substituted by their corresponding LNAunit and wherein said oligonucleotide does not comprise a region of morethan 7 contiguous DNA units.

The high affinity nucleotide analogues are nucleotide analogues whichresult in oligonucleotide which has a higher thermal duplex stabilitywith a complementary RNA nucleotide than the binding affinity of anequivalent DNA nucleotide. This is typically determined by measuring theT_(m).

We have not identified any significant off-target effects when usingthese short, high affinity oligonucleotides targeted against specificmiRNAs. Indeed, the evidence provided herein indicates the effects onmRNA expression are either due to the presence of a complementarysequence to the targeted miRNA (primary mRNA targets) within the mRNA orsecondary effects on mRNAs which are regulated by primary mRNA targets(secondary mRNA targets). No toxicity effects were identified indicatingno significant detrimental off-target effects.

The invention further provides for the use of an oligonucleotideaccording to the invention, such as those which may form part of thepharmaceutical composition, for the manufacture of a medicament for thetreatment of a disease or medical disorder associated with the presenceor over-expression (upregulation) of the microRNA.

The invention further provides for a method for the treatment of adisease or medical disorder associated with the presence orover-expression of the microRNA, comprising the step of administering acomposition (such as the pharmaceutical composition) according to theinvention to a person in need of treatment.

The invention further provides for a method for reducing the effectiveamount of a miRNA in a cell or an organism, comprising administering acomposition (such as the pharmaceutical composition) according to theinvention or a single stranded oligonucleotide according to theinvention to the cell or the organism. Reducing the effective amount inthis context refers to the reduction of functional miRNA present in thecell or organism. It is recognised that the preferred oligonucleotidesaccording to the invention may not always significantly reduce theactual amount of miRNA in the cell or organism as they typically formvery stable duplexes with their miRNA targets.

The invention further provides for a method for de-repression of atarget mRNA of a miRNA in a cell or an organism, comprisingadministering a composition (such as the pharmaceutical composition) ora single stranded oligonucleotide according to the invention to the cellor the organism.

The invention further provides for the use of a single strandedoligonucleotide of between 8-16 such as 8-16 such as between 10-16nucleobases in length, for the manufacture of a medicament for thetreatment of a disease or medical disorder associated with the presenceor over-expression of the microRNA.

The invention further provides for a method for the treatment of adisease or medical disorder associated with the presence orover-expression of the microRNA, comprising the step of administering acomposition (such as the pharmaceutical composition) comprising a singlestranded oligonucleotide of between 8-16 such as between 10-16nucleobases in length to a person in need of treatment.

The invention further provides for a method for reducing the effectiveamount of a miRNA target (i.e. the amount of miRNA which is available torepress target mRNAs) in a cell or an organism, comprising administeringa composition (such as the pharmaceutical composition) comprising asingle stranded oligonucleotide of between 8-16 such as between 10-16nucleobases to the cell or the organism.

The invention further provides for a method for de-repression of atarget mRNA of a miRNA in a cell or an organism, comprising a singlestranded oligonucleotide of between 8-16 such as between 10-16nucleobases or (or a composition comprising said oligonucleotide) to thecell or the organism.

The invention further provides for a method for the synthesis of asingle stranded oligonucleotide targeted against a human microRNAselected from the group consisting of human micro-RNAs miR-19b, miR-21,miR-122A, miR-155 and miR-375, such as a single stranded oligonucleotidedescribed herein, said method comprising the steps of:

-   -   a. Optionally selecting a first nucleobase, counting from the 3′        end, which is a nucleotide analogue, such as an LNA nucleobase.    -   b. Optionally selecting a second nucleobase, counting from the        3′ end, which is an nucleotide analogue, such as an LNA        nucleobase.    -   c. Selecting a region of the single stranded oligonucleotide        which corresponds to the miRNA seed region, wherein said region        is as defined herein.    -   d. Optionally selecting a seventh and eight nucleobase is as        defined herein.    -   e. Optionally selecting between 1 and 10 further nucleobases        which may be selected from the group consisting of        nucleotides (x) and nucleotide analogues (X), such as LNA.    -   f. Optionally selecting a 5′ region of the single stranded        oligonucleotide is as defined herein.    -   g. Optionally selecting a 5′ terminal of the single stranded        oligonucleotide is as defined herein.

Wherein the synthesis is performed by sequential synthesis of theregions defined in steps a-g, wherein said synthesis may be performed ineither the (a to g) or 5′-3′ (g to a) direction, and wherein said singlestranded oligonucleotide is complementary to a sequence of the miRNAtarget.

In one embodiment the oligonucleotide of the invention is designed notto be recruited by RISC or to mediate RISC directed cleavage of themiRNA target. It has been considered that by using longoligonucleotides, e.g. 21 or 22 mers, particularly RNA oligonucleotides,or RNA ‘analogue’ oligonucleotide which are complementary to the miRNAtarget, the oligonucleotide can compete against the target mRNA in termsof RISC complex association, and thereby alleviate miRNA repression ofmiRNA target mRNAs via the introduction of an oligonucleotide whichcompetes as a substrate for the miRNA.

However, the present invention seeks to prevent such undesirable targetmRNA cleavage or translational inhibition by providing oligonucleotidescapable of complementary, and apparently in some cases almostirreversible binding to the mature microRNA. This appears to result in aform of protection against degredation or cleavage (e.g. by RISC orRNAseH or other endo or exo-nucleases), which may not result insubstantial or even significant reduction of the miRNA (e.g. as detectedby northern blot using LNA probes) within a cell, but ensures that theeffective amount of the miRNA, as measured by de-respression analysis isreduced considerably. Therefore, in one aspect, the invention providesoligonucleotides which are purposefully designed not to be compatiblewith the RISC complex, but to remove miRNA by titration by theoligonucleotide. Although not wishing to be bound to a specific theoryof why the oligonucleotides of the present invention are so effective,in analogy with the RNA based oligonucleotides (or complete 2′OMeoligonucleotides), it appears that the oligonucleotides according to thepresent invention work through non-competitive inhibition of miRNAfunction as they effectively remove the available miRNA from thecytoplasm, where as the prior art oligonucleotides provide analternative miRNA substrate, which may act as a competitor inhibitor,the effectiveness of which would be far more dependant upon theconcentration of the oligonucleotide in the cytoplasm, as well as theconcentration of the target mRNA and miRNA.

Whilst not wishing to be bound to any specific theory, one furtherpossibility that may exist with the use of oligonucleotides ofapproximately similar length to the miRNA targets (i.e. the miRNA) isthat the oligonucleotides could form a siRNA like duplex with the miRNAtarget, a situation which would reduce the effectiveness of theoligonucleotide. It is also possible that the oligonucleotidesthemselves could be used as the guiding strand within the RISC complex,thereby generating the possibility of RISC directed degredation ofnon-specific targets which just happen to have sufficientcomplementarity to the oligonucleotide guide.

By selecting short oligonucleotides for targeting miRNA sequences, suchproblems are avoided.

Short oligonucleotides which incorporate LNA are known from the reagentsarea, such as the LNA (see for example WO2005/098029 and WO2006/069584). However the molecules designed for diagnostic or reagentuse are very different in design than those for pharmaceutical use. Forexample, the terminal nucleobases of the reagent oligos are typicallynot LNA, but DNA, and the internucleoside linkages are typically otherthan phosphorothioate, the preferred linkage for use in theoligonucleotides of the present invention. The invention thereforeprovides for a novel class of oligonucleotide per se.

The invention further provides for a (single stranded) oligonucleotideas described in the context of the pharmaceutical composition of theinvention, wherein said oligonucleotide comprises either

-   -   i) at least one phosphorothioate linkage and/or    -   ii) at least one 3′ terminal LNA unit, and/or    -   iii) at least one 5′ terminal LNA unit.

It is preferable for most therapeutic uses that the oligonucleotide isfully phosphorothiolated—the exception being for therapeuticoligonucleotides for use in the CNS, such as in the brain or spine wherephosphorothioation can be toxic, and due to the absence of nucleases,phosphodiester bonds may be used, even between consecutive DNA units. Asreferred to herein, other preferred aspects of the oligonucleotideaccording to the invention is that the second 3′ nucleobase, and/or the9^(th) and 10^(th) (from the 3′ end), may also be LNA.

The inventors have found that other methods of avoiding RNA cleavage(such as exo-nuclease degredation in blood serum, or RISC associatedcleavage of the oligonucleotide according to the invention are possible,and as such the invention also provides for a single strandedoligonucleotide which comprises of either:

-   -   a. an LNA unit at position 1 and 2 counting from the 3′ end        and/or    -   b. an LNA unit at position 9 and/or 10, also counting from the        3′ end, and/or    -   c. either one or two 5′ LNA units.

Whislt the benefits of these other aspects may be seen with longeroligonucleotides, such as nucleotide of up to 26 nucleobase units inlength, it is considered these features may also be used with theshorter oligonucleotides referred to herein, such as theoligonucleotides of between 8-17, 8-16, 10-17 or 10-16 nucleobasesdescribed herein. It is highly preferably that the oligonucleotidescomprise high affinity nucleotide analogues, such as those referred toherein, most preferably LNA units.

The inventors have therefore surprisingly found that carefully designedsingle stranded oligonucleotides comprising locked nucleic acid (LNA)units in a particular order show significant silencing of microRNAs,resulting in reduced microRNA levels. It was found that tight binding ofsaid oligonucleotides to the so-called seed sequence, nucleotides 2 to 8or 2-7, counting from the 5′ end, of the target microRNAs was important.Nucleotide 1 of the target microRNAs is a non-pairing base and is mostlikely hidden in a binding pocket in the Ago 2 protein. Whislt notwishing to be bound to a specific theory, the present inventors considerthat by selecting the seed region sequences, particularly witholigonucleotides that comprise LNA, preferably LNA units in the regionwhich is complementary to the seed region, the duplex between miRNA andoligonucleotide is particularly effective in targeting miRNAs, avoidingoff target effects, and possibly providing a further feature whichprevents RISC directed miRNA function.

The inventors have surprisingly found that microRNA silencing is evenmore enhanced when LNA-modified single stranded oligonucleotides do notcontain a nucleotide at the 3′ end corresponding to this non-pairednucleotide 1. It was further found that two LNA units in the 3′ end ofthe oligonucleotides according to the present invention made saidoligonucleotides highly nuclease resistant.

It was further found that the oligonucleotides of the invention whichhave at least one nucleotide analogue, such as an LNA nucleotide in thepositions corresponding to positions 10 and 11, counting from the 5′end, of the target microRNA may prevent cleavage of the oligonucleotidesof the invention

Accordingly, in one aspect of the invention relates to anoligonucleotide having a length of from 12 to 26 nucleotides, wherein

-   -   i) the first nucleotide, counting from the 3′ end, is a locked        nucleic acid (LNA) unit;    -   ii) the second nucleotide, counting from the 3′ end, is an LNA        unit; and    -   iii) the ninth and/or the tenth nucleotide, counting from the 3′        end, is an LNA unit.

The invention further provides for the oligonucleotides as definedherein for use as a medicament.

The invention further relates to compositions comprising theoligonucleotides defined herein and a pharmaceutically acceptablecarrier.

As mentioned above, microRNAs are related to a number of diseases.Hence, a fourth aspect of the invention relates to the use of anoligonucleotide as defined herein for the manufacture of a medicamentfor the treatment of a disease associated with the expression ofmicroRNAs selected from the group consisting of spinal muscular atrophy,Tourette's syndrome, hepatitis C virus, fragile X mental retardation,DiGeorge syndrome and cancer, such as chronic lymphocytic leukemia,breast cancer, lung cancer and colon cancer, in particular cancer.

A further aspect of the invention is a method to reduce the levels oftarget microRNA by contacting the target microRNA to an oligonucleotideas defined herein, wherein the oligonucleotide

-   -   1. is complementary to the target microRNA    -   2. does not contain a nucleotide at the 3′ end that corresponds        to the first 5′ end nucleotide of the target microRNA.

The invention further provides for an oligonucleotide comprising anucleobase sequence selected from the group consisting of SEQ ID NO 20,SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25,SEQ ID NO 28, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 82, SEQ ID NO 83,SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86, SEQ ID NO 87, SEQ ID NO 88,and SEQ ID NO 89; wherein a lowercase letter identifies the nitrogenousbase of a DNA unit and an uppercase letter identifies the nitrogenousbase of an LNA unit; and wherein the LNA cytosines are optionallymethylated, or a conjugate thereof.

The invention further provides for an oligonucleotide comprising anucleobase sequence selected from the group consisting of SEQ ID NO 11,SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16,SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 90, SEQ ID NO 91,SEQ ID NO 92, SEQ ID NO 93, SEQ ID NO 94, SEQ ID NO 95, SEQ ID NO 96,SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99, SEQ ID NO 100, and SEQ ID NO101; wherein a lowercase letter identifies the nitrogenous base of a DNAunit and an uppercase letter identifies the nitrogenous base of an LNAunit; and wherein the LNA cytosines are optionally methylated, or aconjugate thereof.

The invention further provides for an oligonucleotide comprising anucleobase sequence selected from the group consisting of SEQ ID NO 29,SEQ ID NO 30, SEQ ID NO 31, SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34,SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 102, SEQ ID NO 103,SEQ ID NO 104, and SEQ ID NO 105; wherein a lowercase letter identifiesthe nitrogenous base of a DNA unit and an uppercase letter identifiesthe nitrogenous base of an LNA unit; and wherein the LNA cytosines areoptionally methylated, or a conjugate thereof.

The invention further provides for an oligonucleotide comprising anucleobase sequence selected from the group consisting of SEQ ID NO 47,SEQ ID NO 48, SEQ ID NO 49, SEQ ID NO 50, SEQ ID NO 51, SEQ ID NO 52,SEQ ID NO 53, SEQ ID NO 54, SEQ ID NO 55, SEQ ID NO 106, SEQ ID NO 107,SEQ ID NO 108 and SEQ ID NO 109; wherein a lowercase letter identifiesthe nitrogenous base of a DNA unit and an uppercase letter identifiesthe nitrogenous base of an LNA unit, and wherein the LNA cytosines areoptionally methylated, or a conjugate thereof.

The invention further provides for an oligonucleotide comprising anucleobase sequence selected from the group consisting of SEQ ID NO 65,SEQ ID NO 66, SEQ ID NO 67, SEQ ID NO 68, and SEQ ID NO 69, SEQ ID NO38, SEQ ID NO 39, SEQ ID NO 40, SEQ ID NO 41, SEQ ID NO 42, SEQ ID NO43, SEQ ID NO 44, SEQ ID NO 45, and SEQ ID NO 46, wherein a lowercaseletter identifies the nitrogenous base of a DNA unit and an uppercaseletter identifies the nitrogenous base of an LNA unit, and wherein theLNA cytosines are optionally methylated, or a conjugate thereof.

In one embodiment, the oligonucleotide may have a nucleobase sequence ofbetween 1-17 nucleobases, such as 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17nucleobases, and as such the oligonucleobase in such an embodiment maybe a contiguous subsequence within the oligonucleotides disclosedherein.

The inventors of the present invention have surprisingly found thatantisense oligonucleotides of a certain length comprising a particularcore DNA sequence and locked nucleic acids (LNAs) in said core sequenceexhibit superior microRNA-inhibiting properties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The effect of treatment with different LNA anti-miRoligonucleotides on target nucleic acid expression in the miR-122aexpressing cell line Huh-7. Shown are amounts of miR-122a (arbitraryunits) derived from miR-122a specific qRT-PCR as compared to untreatedcells (mock). The LNA anti-miR oligonucleotides were used at twoconcentrations, 1 and 100 nM, respectively. Included is also a mismatchcontrol (SPC3350) to SPC3349 (also referred to herein as SPC3549).

FIG. 2. Assessment of LNA anti-miR-122a knock-down dose-response forSPC3548 and SPC3549 in comparison with SPC3372 in vivo in mice liversusing miR-122a real-time RT-PCR.

FIG. 2 b miR-122 levels in the mouse liver after treatment withdifferent LNA-antimiRs. The LNA-antimiR molecules SPC3372 and SPC3649were administered into normal mice by three i.p. injections on everysecond day over a six-day-period at indicated doses and sacrificed 48hours after last dose. Total RNA was extracted from the mice livers andmiR-122 was measured by miR-122 specific qPCR.

FIG. 3. Assessment of plasma cholesterol levels in LNA-antimiR-122atreated mice compared to the control mice that received saline.

FIG. 4 a. Assessment of relative Bckdk mRNA levels in LNA antimiR-122atreated mice in comparison with saline control mice using real-timequantitative RT-PCR.

FIG. 4 b. Assessment of relative aldolase A mRNA levels in LNAantimiR-122a treated mice in comparison with saline control mice usingreal-time quantitative RT-PCR.

FIG. 4 c. Assessment of GAPDH mRNA levels in LNA antimiR-122a treatedmice (animals 4-30) in comparison with saline control mice (animals 1-3)using real-time quantitative RT-PCR.

FIG. 5. Assessment of LNA-antimiR™-122a knock-down dose-response in vivoin mice livers using miR-122a real-time RT-PCR. Six groups of animals (5mice per group) were treated in the following manner. Group 1 animalswere injected with 0.2 ml saline by i.v. on 3 successive days, Group 2received 2.5 mg/kg SPC3372, Group 3 received 6.25 mg/kg, Group 4received 12.5 mg/kg and Group 5 received 25 mg/kg, while Group 6received 25 mg/kg SPC3373 (mismatch LNA-antimiR™ oligonucleotide), allin the same manner. The experiment was repeated (therefore n=10) and thecombined results are shown.

FIG. 6. Northern blot comparing SPC3649 with SPC3372. Total RNA from onemouse in each group were subjected to miR-122 specific northern blot.Mature miR-122 and the duplex (blocked microRNA) formed between theLNA-antimiR and miR-122 is indicated.

FIG. 7. Mice were treated with 25 mg/kg/day LNA-antimiR or saline forthree consecutive days and sacrificed 1, 2 or 3 weeks after last dose.Included are also the values from the animals sacrificed 24 hours afterlast dose (example 11 “old design”). miR-122 levels were assessed byqPCR and normalized to the mean of the saline group at each individualtime point. Included are also the values from the animals sacrificed 24hours after last dose (shown mean and SD, n=7, 24 h n=10). Sacrifice day9, 16 or 23 corresponds to sacrifice 1, 2 or 3 weeks after last dose.).

FIG. 8. Mice were treated with 25 mg/kg/day LNA-antimiR or saline forthree consecutive days and sacrificed 1, 2 or 3 weeks after last dose.Included are also the values from the animals sacrificed 24 hours afterlast dose (example 11 “old design”). Plasma cholesterol was measured andnormalized to the saline group at each time point (shown mean and SD,n=7, 24 h n=10).

FIG. 9. Dose dependent miR-122a target mRNA induction by SPC3372inhibition of miR-122a. Mice were treated with different SPC3372 dosesfor three consecutive days, as described above and sacrificed 24 hoursafter last dose. Total RNA extracted from liver was subjected to qPCR.Genes with predicted miR-122 target site and observed to be upregulatedby microarray analysis were investigated for dose-dependent induction byincreasing SPC3372 doses using qPCR. Total liver RNA from 2 to 3 miceper group sacrificed 24 hours after last dose were subjected to qPCR forthe indicated genes. Shown in FIG. 9 is mRNA levels relative to Salinegroup, n=2-3 (2.5-12.5 mg/kg/day: n=2, no SD). Shown is also themismatch control (mm, SPC3373)

FIG. 10. Transient induction of miR-122a target mRNAs following SPC3372treatment. NMRI female mice were treated with 25 mg/kg/day SPC3372 alongwith saline control for three consecutive days and sacrificed 1, 2 or 3weeks after last dose, respectively. RNA was extracted from livers andmRNA levels of predicted miR-122a target mRNAs, selected by microarraydata were investigated by qPCR. Three animals from each group wereanalysed.

FIG. 11. Induction of Vldlr in liver by SPC3372 treatment. The sameliver RNA samples as in previous example (FIG. 10) were investigated forVldlr induction.

FIG. 12. Stability of miR-122a/SPC3372 duplex in mouse plasma. Stabilityof SPC3372 and SPC3372/miR-122a duplex were tested in mouse plasma at37° C. over 96 hours. Shown in FIG. 12 is a SYBR-Gold stained PAGE.

FIG. 13. Sequestering of mature miR-122a by SPC3372 leads to duplexformation. Shown in FIG. 13 is a membrane probed with a miR-122aspecific probe (upper panel) and re-probed with a Let-7 specific probe(lower panel). With the miR-122 probe, two bands could be detected, onecorresponding to mature miR-122 and one corresponding to a duplexbetween SPC3372 and miR-122.

FIG. 14. miR-122a sequestering by SPC3372 along with SPC3372distribution assessed by in situ hybridization of liver sections. Livercryo-sections from treated animals were

FIG. 15. Liver gene expression in miR-122 LNA-antimiR treated mice.Saline and LNA-antimiR treated mice were compared by genome-wideexpression profiling using Affymetrix Mouse Genome 430 2.0 arrays. (a,1)Shown is number of probes displaying differentially expression in liversamples of LNA-antimiR-122 treated and saline treated mice 24 hours posttreatment. (b,2) The occurrence of miR-122 seed sequence indifferentially expressed genes. The plot shows the percentage oftranscripts with at least one miR-122 seed recognition sequence in their3′ UTR. Random: Random sequences were generated and searched for miR-122seed recognition sequences. Temporal liver gene expression profiles inLNA-antimiR treated mice. Mice were treated with 25 mg/kg/dayLNA-antimiR or saline for three consecutive days and sacrificed 1, 2 or3 weeks after last dose. Included are also the values from the animalssacrificed 24 hours after last dose. (c,3) RNA samples from differenttime points were also subjected to expression profiling. Hierarchicalcluster analysis of expression profiles of genes identified asdifferentially expressed between LNA-antimiR and saline treated mice 24hours, one week or three weeks post treatment. (d,4) Expression profilesof genes identified as differentially expressed between LNA-antimiR andsaline treated mice 24 hours post treatment were followed over time. Theexpression ratios of up- and down-regulated genes in LNA-antimiR treatedmice approach 1 over the time-course, indicating a reversible effect ofthe LNA-antimiR treatment.

FIG. 16. The effect of treatment with SPC3372 and 3595 on miR-122 levelsin mice livers.

FIG. 17. The effect of treatment with SPC3372 and 3595 on Aldolase Alevels in mice livers.

FIG. 18. The effect of treatment with SPC3372 and 3595 on Bckdk levelsin mice livers.

FIG. 19. The effect of treatment with SPC3372 and 3595 on CD320 levelsin mice livers.

FIG. 20. The effect of treatment with SPC3372 and 3595 on Ndrg3 levelsin mice livers.

FIG. 21. The effect of long-term treatment with SPC3649 on total plasmacholesterol in hypercholesterolemic and normal mice. Weekly samples ofblood plasma were obtained from the SPC3649 treated and saline controlmice once weekly followed by assessment of total plasma cholesterol. Themice were treated with 5 mg/kg SPC3649, SPC3744 or saline twice weekly.Normal mice given were treated in parallel.

FIG. 22. The effect of long-term treatment with SPC3649 on miR-122levels in hypercholesterolemic and normal mice.

FIG. 23. The effect of long-term treatment with SPC3649 on Aldolase Alevels in hypercholesterolemic and normal mice.

FIG. 24. The effect of long-term treatment with SPC3649 on Bckdk levelsin hypercholesterolemic and normal mice.

FIG. 25. The effect of long-term treatment with SPC3649 on AST levels inhypercholesterolemic and normal mice.

FIG. 26. The effect of long-term treatment with SPC3649 on ALT levels inhypercholesterolemic and normal mice.

FIG. 27. Modulation of HCV replication by SPC3649 in a Huh-7 cell model.Northern blot analysis of HCV RNA in Huh-7 cells after transfection withdifferent LNA-antimiR (SPC3648, SPC3649 and SPC3550) and 2′OMeantago-mir-122 molecules (upper panel). The hybridisation signalintensities were quantified and normalized to spectrin mRNA signals ineach lane (lower panel).

FIG. 28. Functional de-repression of renilla luciferase with miR-19btarget by miR-19b blocking oligonucleotides in an endogenously miR-19bexpressing cell line, HeLa. “miR-19b target” is the plasmid with miR-19btarget but not co-trasfected with oligo blocking miR-19b and hencerepresent fully miR-19b repressed renilla luciferase expression.

FIG. 29. Functional de-repression of renilla luciferase with miR-122target by miR-122 blocking oligonucleotides in an endogenously miR-122expressing cell line, Huh-7. “miR-122 target” is the correspondingplasmid with miR-122 target but not co-trasfected with oligo blockingmiR-122 and hence represent fully miR-122 repressed renilla luciferaseexpression.

FIG. 30. Diagram illustrating the alignment of an oligonucleotideaccording to the invention and a microRNA target.

DETAILED DESCRIPTION OF THE INVENTION

The oligonucleotide of the invention is typically single stranded. Itwill therefore be understood that within the context of the inventionthe term oligonucleotide may be used interchangeably with the termsingle stranded oligonucleotide.

In one embodiment, the invention provides pharmaceutical compositionscomprising short (single stranded) oligonucleotides, of length ofbetween 8 and 17 nucleobases in length, such as between 10 and 17nucleobases which are complementary to human microRNAs. The shortoligonucleotides are particularly effective at alleviating miRNArepression in vivo. It is found that the incorporation of high affinitynucleotide analogues into the oligonucleotides results in highlyeffective anti-microRNA molecules which appear to function via theformation of almost irreversible duplexes with the miRNA target, ratherthan RNA cleavage based mechanisms, such as mechanisms associated withRNaseH or RISC.

It is highly preferable that the single stranded oligonucleotideaccording to the invention comprises a region of contiguous nucleobasesequence which is 100% complementary to the human microRNA seed region.

It is preferable that single stranded oligonucleotide according to theinvention is complementary to the mature human microRNA sequence.

Preferred oligonucleotides according to the invention are complementaryto a microRNA sequence selected from the group consisting of has-miR19b,hsa-miR21, hsa-miR 122, hsa-miR 142 a7b, hsa-miR 155, hsa-miR 375.

In one embodiment, the oligonucleotide according to the invention doesnot comprise a nucleobase at the 3′ end that corresponds to the first 5′end nucleotide of the target microRNA.

In one embodiment, the first nucleobase of the single strandedoligonucleotide according to the invention, counting from the 3′ end, isa nucleotide analogue, such as an LNA unit.

In one embodiment, the second nucleobase of the single strandedoligonucleotide according to the invention, counting from the 3′ end, isa nucleotide analogue, such as an LNA unit.

In one embodiment, the ninth and/or the tenth nucleotide of the singlestranded oligonucleotide according to the invention, counting from the3′ end, is a nucleotide analogue, such as an LNA unit.

In one embodiment, the ninth nucleobase of the single strandedoligonucleotide according to the invention, counting from the 3′ end isa nucleotide analogue, such as an LNA unit.

In one embodiment, the tenth nucleobase of the single strandedoligonucleotide according to the invention, counting from the 3′ end isa nucleotide analogue, such as an LNA unit.

In one embodiment, both the ninth and the tenth nucleobase of the singlestranded oligonucleotide according to the invention, calculated from the3′ end is a nucleotide analogue, such as an LNA unit.

In one embodiment, the single stranded oligonucleotide according to theinvention does not comprise a region of more than 5 consecutive DNAnucleotide units. In one embodiment, the single stranded oligonucleotideaccording to the invention does not comprise a region of more than 6consecutive DNA nucleotide units. In one embodiment, the single strandedoligonucleotide according to the invention does not comprise a region ofmore than 7 consecutive DNA nucleotide units. In one embodiment, thesingle stranded oligonucleotide according to the invention does notcomprise a region of more than 8 consecutive DNA nucleotide units. Inone embodiment, the single stranded oligonucleotide according to theinvention does not comprise a region of more than 3 consecutive DNAnucleotide units. In one embodiment, the single stranded oligonucleotideaccording to the invention does not comprise a region of more than 2consecutive DNA nucleotide units.

In one embodiment, the single stranded oligonucleotide comprises atleast region consisting of at least two consecutive nucleotide analogueunits, such as at least two consecutive LNA units.

In one embodiment, the single stranded oligonucleotide comprises atleast region consisting of at least three consecutive nucleotideanalogue units, such as at least three consecutive LNA units.

In one embodiment, the single stranded oligonucleotide of the inventiondoes not comprise a region of more than 7 consecutive nucleotideanalogue units, such as LNA units. In one embodiment, the singlestranded oligonucleotide of the invention does not comprise a region ofmore than 6 consecutive nucleotide analogue units, such as LNA units. Inone embodiment, the single stranded oligonucleotide of the inventiondoes not comprise a region of more than 5 consecutive nucleotideanalogue units, such as LNA units. In one embodiment, the singlestranded oligonucleotide of the invention does not comprise a region ofmore than 4 consecutive nucleotide analogue units, such as LNA units. Inone embodiment, the single stranded oligonucleotide of the inventiondoes not comprise a region of more than 3 consecutive nucleotideanalogue units, such as LNA units. In one embodiment, the singlestranded oligonucleotide of the invention does not comprise a region ofmore than 2 consecutive nucleotide analogue units, such as LNA units.

In one embodiment, the first or second 3′ nucleobase of the singlestranded oligonucleotide corresponds to the second 5′ nucleotide of themicroRNA sequence.

In one embodiment, nucleobase units 1 to 6 (inclusive) of the singlestranded oligonucleotide as measured from the 3′ end the region of thesingle stranded oligonucleotide are complementary to the microRNA seedregion sequence.

In one embodiment, nucleobase units 1 to 7 (inclusive) of the singlestranded oligonucleotide as measured from the 3′ end the region of thesingle stranded oligonucleotide are complementary to the microRNA seedregion sequence.

In one embodiment, nucleobase units 2 to 7 (inclusive) of the singlestranded oligonucleotide as measured from the 3′ end the region of thesingle stranded oligonucleotide are complementary to the microRNA seedregion sequence.

In one embodiment, the single stranded oligonucleotide comprises atleast one nucleotide analogue unit, such as at least one LNA unit, in aposition which is within the region complementary to the miRNA seedregion. The single stranded oligonucleotide may, in one embodimentcomprise at between one and 6 or between 1 and 7 nucleotide analogueunits, such as between 1 and 6 and 1 and 7 LNA units, in a positionwhich is within the region complementary to the miRNA seed region.

In one embodiment, the nucleobase sequence of the single strandedoligonucleotide which is complementary to the sequence of the microRNAseed region, is selected from the group consisting of (X)Xxxxxx,(X)xXxxxx, (X)xxXxxx, (X)xxxXxx, (X)xxxxXx and (X)xxxxxX, as read in a3′-5′direction, wherein “X” denotes a nucleotide analogue, (X) denotesan optional nucleotide analogue, such as an LNA unit, and “x” denotes aDNA or RNA nucleotide unit.

In one embodiment, the single stranded oligonucleotide comprises atleast two nucleotide analogue units, such as at least two LNA units, inpositions which are complementary to the miRNA seed region.

In one embodiment, the nucleobase sequence of the single strandedoligonucleotide which is complementary to the sequence of the microRNAseed region, is selected from the group consisting of (X)XXxxxx,(X)XxXxxx, (X)XxxXxx, (X)XxxxXx, (X)XxxxxX, (X)xXXxxx, (X)xXxXxx,(X)xXxxXx, (X)xXxxxX, (X)xxXXxx, (X)xxXxXx, (X)xxXxxX, (X)xxxXXx,(X)xxxXxX and (X)xxxxXX, wherein “X” denotes a nucleotide analogue, suchas an LNA unit, (X) denotes an optional nucleotide analogue, such as anLNA unit, and “x” denotes a DNA or RNA nucleotide unit.

In one embodiment, the single stranded oligonucleotide comprises atleast three nucleotide analogue units, such as at least three LNA units,in positions which are complementary to the miRNA seed region.

In one embodiment, the nucleobase sequence of the single strandedoligonucleotide which is complementary to the sequence of the microRNAseed region, is selected from the group consisting of (X)XXXxxx,(X)xXXXxx, (X)xxXXXx, (X)xxxXXX, (X)XXxXxx, (X)XXxxXx, (X)XXxxxX,(X)xXXxXx, (X)xXXxxX, (X)xxXXxX, (X)XxXXxx, (X)XxxXXx, (X)XxxxXX,(X)xXxXXx, (X)xXxxXX, (X)xxXxXX, (X)xXxXxX and (X)XxXxXx, wherein “X”denotes a nucleotide analogue, such as an LNA unit, (X) denotes anoptional nucleotide analogue, such as an LNA unit, and “x” denotes a DNAor RNA nucleotide unit.

In one embodiment, the single stranded oligonucleotide comprises atleast four nucleotide analogue units, such as at least four LNA units,in positions which are complementary to the miRNA seed region.

In one embodiment the nucleobase sequence of the single strandedoligonucleotide which is complementary to the sequence of the microRNAseed region, is selected from the group consisting of (X)xxXXX,(X)xXxXXX, (X)xXXxXX, (X)xXXXxX, (X)xXXXXx, (X)XxxXXXX, (X)XxXxXX,(X)XxXXxX, (X)XxXXx, (X)XXxxXX, (X)XXxXxX, (X)XXxXXx, (X)XXXxxX,(X)XXXxXx, and (X)XXXXxx, wherein “X” denotes a nucleotide analogue,such as an LNA unit, (X) denotes an optional nucleotide analogue, suchas an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.

In one embodiment, the single stranded oligonucleotide comprises atleast five nucleotide analogue units, such as at least five LNA units,in positions which are complementary to the miRNA seed region.

In one embodiment, the nucleobase sequence of the single strandedoligonucleotide which is complementary to the sequence of the microRNAseed region, is selected from the group consisting of (X)xXXXXX,(X)XxXXXX, (X)XXxXXX, (X)XXXxXX, (X)XXXXxX and (X)XXXXXx, wherein “X”denotes a nucleotide analogue, such as an LNA unit, (X) denotes anoptional nucleotide analogue, such as an LNA unit, and “x” denotes a DNAor RNA nucleotide unit.

In one embodiment, the single stranded oligonucleotide comprises six orseven nucleotide analogue units, such as six or seven LNA units, inpositions which are complementary to the miRNA seed region.

In one embodiment, the nucleobase sequence of the single strandedoligonucleotide which is complementary to the sequence of the microRNAseed region, is selected from the group consisting of XXXXXX, XxXXXXX,XXxXXXX, XXXxXXX, XXXXxXX, XXXXXxX and XXXXXXx, wherein “X” denotes anucleotide analogue, such as an LNA unit, such as an LNA unit, and “x”denotes a DNA or RNA nucleotide unit.

In one embodiment, the two nucleobase motif at position 7 to 8, countingfrom the 3′ end of the single stranded oligonucleotide is selected fromthe group consisting of xx, XX, xX and Xx, wherein “X” denotes anucleotide analogue, such as an LNA unit, such as an LNA unit, and “x”denotes a DNA or RNA nucleotide unit.

In one embodiment, the two nucleobase motif at position 7 to 8, countingfrom the 3′ end of the single stranded oligonucleotide is selected fromthe group consisting of XX, xX and Xx, wherein “X” denotes a nucleotideanalogue, such as an LNA unit, such as an LNA unit, and “x” denotes aDNA or RNA nucleotide unit.

In one embodiment, the single stranded oligonucleotide comprises atleast 12 nucleobases and wherein the two nucleobase motif at position 11to 12, counting from the 3′ end of the single stranded oligonucleotideis selected from the group consisting of xx, XX, xX and Xx, wherein “X”denotes a nucleotide analogue, such as an LNA unit, such as an LNA unit,and “x” denotes a DNA or RNA nucleotide unit.

In one embodiment, the single stranded oligonucleotide comprises atleast 12 nucleobases and wherein the two nucleobase motif at position 11to 12, counting from the 3′ end of the single stranded oligonucleotideis selected from the group consisting of XX, xX and Xx, wherein “X”denotes a nucleotide analogue, such as an LNA unit, such as an LNA unit,and “x” denotes a DNA or RNA nucleotide unit.

In one embodiment, the single stranded oligonucleotide comprises atleast 13 nucleobases and wherein the three nucleobase motif at position11 to 13, counting from the 3′ end, is selected from the groupconsisting of xxx, Xxx, xXx, xxX, XXx, XxX, xXX and XXX, wherein “X”denotes a nucleotide analogue, such as an LNA unit, such as an LNA unit,and “x” denotes a DNA or RNA nucleotide unit.

In one embodiment, the three nucleobase motif at position 11 to 13,counting from the 3′ end of the single stranded oligonucleotide, isselected from the group consisting of Xxx, xXx, xxX, XXx, XxX, xXX andXXX, wherein “X” denotes a nucleotide analogue, such as an LNA unit,such as an LNA unit, and “x” denotes a DNA or RNA nucleotide unit.

In one embodiment, the single stranded oligonucleotide comprises atleast 14 nucleobases and wherein the four nucleobase motif at positions11 to 14, counting from the 3′ end, is selected from the groupconsisting of xxxx, Xxxx, xXxx, xxXx, xxxX, XXxx, XxXx, XxxX, xXXx,xXxX, xxXX, XXXx, XxXX, xXXX, XXxX and XXXX wherein “X” denotes anucleotide analogue, such as an LNA unit, such as an LNA unit, and “x”denotes a DNA or RNA nucleotide unit.

In one embodiment, the four nucleobase motif at position 11 to 14 of thesingle stranded oligonucleotide, counting from the 3′ end, is selectedfrom the group consisting of Xxxx, xXxx, xxXx, xxxX, XXxx, XxXx, XxxX,xXXx, xXxX, xxXX, XXXx, XxXX, xXXX, XXxX and XXXX, wherein “X” denotes anucleotide analogue, such as an LNA unit, and “x” denotes a DNA or RNAnucleotide unit.

In one embodiment, the single stranded oligonucleotide comprises 15nucleobases and the five nucleobase motif at position 11 to 15, countingfrom the 3′ end, is selected from the group consisting of Xxxxx, xXxxx,xxXxx, xxxXx, xxxxX, XXxxx, XxXxx, XxxXx, XxxxX, xXXxx, xXxXx, xXxxX,xxXXx, xxXxX, xxxXX, XXXxx, XXxxX, XxxXX, xXXXx, xxXXX, XXxXX, XxXxX,XXXXx, XXXxX, XXxXX, XxXXXX, xXXXX, and XXXXX wherein “X” denotes anucleotide analogue, such as an LNA unit, such as an LNA unit, and “x”denotes a DNA or RNA nucleotide unit.

In one embodiment, the single stranded oligonucleotide comprises 16nucleobases and the six nucleobase motif at positions 11 to 16, countingfrom the 3′ end, is selected from the group consisting of Xxxxxx,xXxxxx, xxXxxx, xxxXxx, xxxxXx, xxxxxX, XXxxxx, XxXxxx, XxxXxx, XxxxXx,XxxxxX, xXXxxx, xXxXxx, xXxxXx, xXxxxX, xxXXxx, xxXxXx, xxXxxX, xxxXXx,xxxXxX, xxxxXX, XXXxxx, XXxXxx, XXxxXx, XXxxxX, XxXXxx, XxXxXx, XxXxxX,XxxXXx, XxxXxX, XxxxXX, xXXXxx, xXXxXx, xXXxxX, xXxXXx, xXxXxX, xXxxXX,xxXXXx, xxXXxX, xxXxXX, xxxXXX, XXXXxx, XXXxxX, XXxxXX, XxxXXX, xxXXXX,xXxXXX, XxXxXX, XXxXxX, XXXxXx, xXXxXX, XxXXxX, XXxXXx, xXXXxX, XxXXXx,xXXXXx, xXXXXX, XxXXXX, XXxXXX, XXXxXX, XXXXxX, XXXXXx, and XXXXXXwherein “X” denotes a nucleotide analogue, such as an LNA unit, such asan LNA unit, and “x” denotes a DNA or RNA nucleotide unit.

In one embodiment, the six nucleobase motif at positions 11 to 16 of thesingle stranded oligonucleotide, counting from the 3′ end, is xxXxxX,wherein “X” denotes a nucleotide analogue, such as an LNA unit, such asan LNA unit, and “x” denotes a DNA or RNA nucleotide unit.

In one embodiment, the three 5′ most nucleobases, is selected from thegroup consisting of Xxx, xXx, xxX, XXx, XxX, xXX and XXX, wherein “X”denotes a nucleotide analogue, such as an LNA unit, such as an LNA unit,and “x” denotes a DNA or RNA nucleotide unit.

In one embodiment, x″ denotes a DNA unit.

In one embodiment, the single stranded oligonucleotide comprises anucleotide analogue unit, such as an LNA unit, at the 5′ end.

In one embodiment, the nucleotide analogue units, such as X, areindependently selected form the group consisting of: 2′-O-alkyl-RNAunit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA unit,PNA unit, HNA unit, INA unit.

In one embodiment, all the nucleobases of the single strandedoligonucleotide of the invention are nucleotide analogue units.

In one embodiment, the nucleotide analogue units, such as X, areindependently selected form the group consisting of: 2′-OMe-RNA units,2′-fluoro-DNA units, and LNA units,

In one embodiment, the single stranded oligonucleotide comprises said atleast one LNA analogue unit and at least one further nucleotide analogueunit other than LNA.

In one embodiment, the non-LNA nucleotide analogue unit or units areindependently selected from 2′-OMe RNA units and 2′-fluoro DNA units.

In one embodiment, the single stranded oligonucleotide consists of atleast one sequence XYX or YXY, wherein X is LNA and Y is either a 2′-OMeRNA unit and 2′-fluoro DNA unit.

In one embodiment, the sequence of nucleobases of the single strandedoligonucleotide consists of alternative X and Y units.

In one embodiment, the single stranded oligonucleotide comprisesalternating LNA and DNA units (Xx) or (xX).

In one embodiment, the single stranded oligonucleotide comprises a motifof alternating LNA followed by 2 DNA units (Xxx), xXx or xxX.

In one embodiment, at least one of the DNA or non-LNA nucleotideanalogue units are replaced with a LNA nucleobase in a position selectedfrom the positions identified as LNA nucleobase units in any one of theembodiments referred to above.

In one embodiment, “X” donates an LNA unit.

In one embodiment, the single stranded oligonucleotide comprises atleast 2 nucleotide analogue units, such as at least 3 nucleotideanalogue units, such as at least 4 nucleotide analogue units, such as atleast 5 nucleotide analogue units, such as at least 6 nucleotideanalogue units, such as at least 7 nucleotide analogue units, such as atleast 8 nucleotide analogue units, such as at least 9 nucleotideanalogue units, such as at least 10 nucleotide analogue units.

In one embodiment, the single stranded oligonucleotide comprises atleast 2 LNA units, such as at least 3 LNA units, such as at least 4 LNAunits, such as at least 5 LNA units, such as at least 6 LNA units, suchas at least 7 LNA units, such as at least 8 LNA units, such as at least9 LNA units, such as at least 10 LNA units.

In one embodiment wherein at least one of the nucleotide analogues, suchas LNA units, is either cytosine or guanine, such as between 1-10 of theof the nucleotide analogues, such as LNA units, is either cytosine orguanine, such as 2, 3, 4, 5, 6, 7, 8, or 9 of the of the nucleotideanalogues, such as LNA units, is either cytosine or guanine.

In one embodiment at least two of the nucleotide analogues such as LNAunits is either cytosine or guanine. In one embodiment at least three ofthe nucleotide analogues such as LNA units is either cytosine orguanine. In one embodiment at least four of the nucleotide analoguessuch as LNA units is either cytosine or guanine. In one embodiment atleast five of the nucleotide analogues such as LNA units is eithercytosine or guanine. In one embodiment at least six of the nucleotideanalogues such as LNA units is either cytosine or guanine. In oneembodiment at least seven of the nucleotide analogues such as LNA unitsis either cytosine or guanine. In one embodiment at least eight of thenucleotide analogues such as LNA units is either cytosine or guanine.

In a preferred embodiment the nucleotide analogues have a higher thermalduplex stability a complementary RNA nucleotide than the bindingaffinity of an equivalent DNA nucleotide to said complementary RNAnucleotide.

In one embodiment, the nucleotide analogues confer enhanced serumstability to the single stranded oligonucleotide.

In one embodiment, the single stranded oligonucleotide forms an A-helixconformation with a complementary single stranded RNA molecule.

A duplex between two RNA molecules typically exists in an A-formconformation, where as a duplex between two DNA molecules typicallyexits in a B-form conformation. A duplex between a DNA and RNA moleculetypically exists in a intermediate conformation (A/B form). The use ofnucleotide analogues, such as beta-D-oxy LNA can be used to promote amore A form like conformation. Standard circular dichromisms (CD) or NMRanalysis is used to determine the form of duplexes between theoligonucleotides of the invention and complementary RNA molecules.

As recruitment by the RISC complex is thought to be dependant upon thespecific structural conformation of the miRNA/mRNA target, theoligonucleotides according to the present invention may, in oneembodiment form a A/B-form duplex with a complementary RNA molecule.

However, we have also determined that the use of nucleotide analogueswhich promote the A-form structure can also be effective, such as thealpha-L isomer of LNA.

In one embodiment, the single stranded oligonucleotide forms an A/B-formconformation with a complementary single stranded RNA molecule.

In one embodiment, the single stranded oligonucleotide forms an A-fromconformation with a complementary single stranded RNA molecule.

In one embodiment, the single stranded oligonucleotide according to theinvention does not mediate RNAseH based cleavage of a complementarysingle stranded RNA molecule. Typically a stretch of at least 5(typically not effective ofr RNAse H recruitment), more preferably atleast 6, more preferably at least 7 or 8 consecutive DNA nucleobases (oralternative nucleobases which can recruit RNAseH, such as alpha L-aminoLNA) are required in order for an oligonucleotide to be effective inrecruitment of RNAseH.

EP 1 222 309 provides in vitro methods for determining RNaseH activity,which may be used to determine the ability to recruit RNaseH. A compoundis deemed capable of recruiting RNase H if, when provided with thecomplementary RNA target, it has an initial rate, as measured inpmol/l/min, of at least 1%, such as at least 5%, such as at least 10% orless than 20% of the equivalent DNA only oligonucleotide, with no 2′substitutions, with phosphorothiote linkage groups between allnucleotides in the oligonucleotide, using the methodology provided byExample 91-95 of EP 1 222 309.

A compound is deemed essentially incapable of recruiting RNaseH if, whenprovided with the complementary RNA target, and RNaseH, the RNaseHinitial rate, as measured in pmol/l/min, is less than 1%, such as lessthan 5%,such as less than 10% or less than 20% of the initial ratedetermined using the equivalent DNA only oligonucleotide, with no 2′substitutions, with phosphiothiote linkage groups between allnucleotides in the oligonucleotide, using the methodology provided byExample 91-95 of EP 1 222 309.

In a highly preferred embodiment, the single stranded oligonucleotide ofthe invention is capable of forming a duplex with a complementary singlestranded RNA nucleic acid molecule (typically of about the same lengthof said single stranded oligonucleotide) with phosphodiesterinternucleoside linkages, wherein the duplex has a T_(m) of at leastabout 60° C., indeed it is preferred that the single strandedoligonucleotide is capable of forming a duplex with a complementarysingle stranded RNA nucleic acid molecule with phosphodiesterinternucleoside linkages, wherein the duplex has a T_(m) of betweenabout 70° C. to about 95° C., such as a T_(m) of between about 70° C. toabout 90° C., such as between about 70° C. and about 85° C.

In one embodiment, the single stranded oligonucleotide is capable offorming a duplex with a complementary single stranded DNA nucleic acidmolecule with phosphodiester internucleoside linkages, wherein theduplex has a T_(m) of between about 50° C. to about 95° C., such asbetween about 50° C. to about 90° C., such as at least about 55° C.,such as at least about 60° C., or no more than about 95° C.

The single stranded oligonucleotide may, in one embodiment have a lengthof between 14-16 nucleobases, including 15 nucleobases.

In one embodiment, the LNA unit or units are independently selected fromthe group consisting of oxy-LNA, thio-LNA, and amino-LNA, in either ofthe D-β and L-α configurations or combinations thereof.

In one specific embodiment the LNA units may be an ENA nucleobase.

In one the embodiment the LNA units are beta D oxy-LNA.

In one embodiment the LNA units are in alpha-L amino LNA.

In a preferable embodiment, the single stranded oligonucleotidecomprises between 3 and 17 LNA units.

In one embodiment, the single stranded oligonucleotide comprises atleast one internucleoside linkage group which differs from phosphate.

In one embodiment, the single stranded oligonucleotide comprises atleast one phosphorothioate internucleoside linkage.

In one embodiment, the single stranded oligonucleotide comprisesphosphodiester and phosphorothioate linkages.

In one embodiment, the all the internucleoside linkages arephosphorothioate linkages.

In one embodiment, the single stranded oligonucleotide comprises atleast one phosphodiester internucleoside linkage.

In one embodiment, all the internucleoside linkages of the singlestranded oligonucleotide of the invention are phosphodiester linkages.

In one embodiment, pharmaceutical composition according to the inventioncomprises a carrier such as saline or buffered saline.

In one embodiment, the method for the synthesis of a single strandedoligonucleotide targeted against a human microRNA, is performed in the3′ to 5′ direction a-f.

The method for the synthesis of the single stranded oligonucleotideaccording to the invention may be performed using standard solid phaseoligonucleotide synthesis.

Further embodiments of the invention, which may be combined with theabove embodiments are shown in the claims and under the title ‘Furtherembodiments’.

Definitions

The term ‘nucleobase’ refers to nucleotides, such as DNA and RNA, andnucleotide analogues.

The term “oligonucleotide” (or simply “oligo”) refers, in the context ofthe present invention, to a molecule formed by covalent linkage of twoor more nucleobases. When used in the context of the oligonucleotide ofthe invention (also referred to the single stranded oligonucleotide),the term “oligonucleotide” may have, in one embodiment, for examplebetween 8-26 nucleobases, such between 10 to 26 nucleobases suchbetween12 to 26 nucleobases. In a preferable embodiment, as detailedherein, the oligonucleotide of the invention has a length of between8-17 nucleobases, such as between 20-27 nucleobases such as between 8-16nucleobases, such as between 12-15 nucleobases,

In such an embodiment, the oligonucleotide of the invention may have alength of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25 or 26 nucleobases.

It will be recognised that for shorter oligonucleotides it may benecessary to increase the proportion of (high affinity) nucleotideanalogues, such as LNA. Therefore in one embodiment at least about 30%of the nucleobases are nucleotide analogues, such as at least about 33%,such as at least about 40%, or at least about 50% or at least about 60%,such as at least about 66%, such as at least about 70%, such as at leastabout 80%, or at least about 90%. It will also be apparent that theoligonucleotide may comprise of a nucleobase sequence which consists ofonly nucleotide analogue sequences.

Herein, the term “nitrogenous base” is intended to cover purines andpyrimidines, such as the DNA nucleobases A, C, T and G, the RNAnucleobases A, C, U and G, as well as non-DNA/RNA nucleobases, such as5-methylcytosine (^(Me)C), isocytosine, pseudoisocytosine,5-bromouracil, 5-propynyluracil, 5-propyny-6-fluoroluracil,5-methylthiazoleuracil, 6-aminopurine, 2-aminopurine, inosine,2,6-diaminopurine, 7-propyne-7-deazaadenine, 7-propyne-7-deazaguanineand 2-chloro-6-aminopurine, in particular ^(Me)C. It will be understoodthat the actual selection of the non-DNA/RNA nucleobase will depend onthe corresponding (or matching) nucleotide present in the microRNAstrand which the oligonucleotide is intended to target. For example, incase the corresponding nucleotide is G it will normally be necessary toselect a non-DNA/RNA nucleobase which is capable of establishinghydrogen bonds to G. In this specific case, where the correspondingnucleotide is G, a typical example of a preferred non-DNA/RNA nucleobaseis ^(Me)C.

The term “internucleoside linkage group” is intended to mean a groupcapable of covalently coupling together two nucleobases, such as betweenDNA units, between DNA units and nucleotide analogues, between twonon-LNA units, between a non-LNA unit and an LNA unit, and between twoLNA units, etc. Preferred examples include phosphate, phpshodiestergroups and phosphorothioate groups.

The internucleoside linkage may be selected form the group consistingof: —O—P(O)₂—O—, —O—P(O,S)—O—, —O—P(S)₂—O—, —S—P(O)₂—O—, —S—P(O,S)—O—,—S—P(S)₂—O—, —O—P(O)₂—S—, —O—P(O,S)—S—, —S—P(O)₂—S—, —O—PO(R^(H))—O—,O—PO(OCH₃)—O—, —O—PO(NR^(H))—O—, —O—PO(OCH₂CH₂S—R)—O—, —O—PO(BH₃)—O—,—O—PO(NHR^(H))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—, —NR^(H)—CO—O—,—NR^(H)—CO—NR^(H)—, and/or the internucleoside linkage may be selectedform the group consisting of: —O—CO—O—, —O—CO—NR^(H)—, —NR^(H)—CO—CH₂—,—O—CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—, —CO—NR^(H)—CH₂—, —CH₂—NR^(H)—CO—,—O—CH₂—CH₂—S—, —S—CH₂—CH₂—O—, —S—CH₂—CH₂—S—, —CH₂—SO₂—CH₂—,—CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—CO—, —CH₂—NCH₃—O—CH₂—, where R^(H) isselected from hydrogen and C₁₋₄-alkyl. Suitably, in some embodiments,sulphur (S) containing internucleoside linkages as provided above may bepreferred

The terms “corresponding to” and “corresponds to” as used in the contextof oligonucleotides refers to the comparison between either a nucleobasesequence of the compound of the invention, and the reverse complementthereof, or in one embodiment between a nucleobase sequence and anequivalent (identical) nucleobase sequence which may for examplecomprise other nucleobases but retains the same base sequence, orcomplement thereof. Nucleotide analogues are compared directly to theirequivalent or corresponding natural nucleotides. Sequences which formthe reverse complement of a sequence are referred to as the complementsequence of the sequence.

When referring to the length of a nucleotide molecule as referred toherein, the length corresponds to the number of monomer units, i.e.nucleobases, irrespective as to whether those monomer units arenucleotides or nucleotide analogues. With respect to nucleobases, theterms monomer and unit are used interchangeably herein.

It should be understood that when the term “about” is used in thecontext of specific values or ranges of values, the disclosure should beread as to include the specific value or range referred to.

Preferred DNA analogues includes DNA analogues where the 2′-H group issubstituted with a substitution other than —OH (RNA) e.g. bysubstitution with —O—CH₃, —O—CH₂—CH₂—O—CH₃, —O—CH₂—CH₂—CH₂—NH₂,—O—CH₂—CH₂—CH₂—OH or —F.

Preferred RNA analogues includes RNA analogues which have been modifiedin its 2′-OH group, e.g. by substitution with a group other than —H(DNA), for example —O—CH₃, —O—CH₂—CH₂—O—CH₃, —O—CH₂—CH₂—CH₂—NH₂,—O—CH₂—CH₂—CH₂—OH or —F.

In one embodiment the nucleotide analogue is “ENA”.

When used in the present context, the terms “LNA unit”, “LNA monomer”,“LNA residue”, “locked nucleic acid unit”, “locked nucleic acid monomer”or “locked nucleic acid residue”, refer to a bicyclic nucleosideanalogue. LNA units are described in inter alia WO 99/14226, WO00/56746, WO 00/56748, WO 01/25248, WO 02/28875, WO 03/006475 and WO03/095467. The LNA unit may also be defined with respect to its chemicalformula. Thus, an “LNA unit”, as used herein, has the chemical structureshown in Scheme 1 below:

wherein

-   -   X is selected from the group consisting of O, S and NR^(H),        where R^(H) is H or C₁₋₄-alkyl;    -   Y is (—CH₂)_(r), where r is an integer of 1-4; and    -   B is a nitrogenous base.

When referring to substituting a DNA unit by its corresponding LNA unitin the context of the present invention, the term “corresponding LNAunit” is intended to mean that the DNA unit has been replaced by an LNAunit containing the same nitrogenous base as the DNA unit that it hasreplaced, e.g. the corresponding LNA unit of a DNA unit containing thenitrogenous base A also contains the nitrogenous base A. The exceptionis that when a DNA unit contains the base C, the corresponding LNA unitmay contain the base C or the base ^(Me)C, preferably ^(Me)C.

Herein, the term “non-LNA unit” refers to a nucleoside different from anLNA-unit, i.e. the term “non-LNA unit” includes a DNA unit as well as anRNA unit. A preferred non-LNA unit is a DNA unit.

The terms “unit”, “residue” and “monomer” are used interchangeablyherein.

In the present context the term “conjugate” is intended to indicate aheterogenous molecule formed by the covalent attachment of anoligonucleotide as described herein to one or more non-nucleotide ornon-polynucleotide moieties. Examples of non-nucleotide ornon-polynucleotide moieties include macromolecular agents such asproteins, fatty acid chains, sugar residues, glycoproteins, polymers, orcombinations thereof. Typically proteins may be antibodies for a targetprotein. Typical polymers may be polyethelene glycol.

The term “at least one” encompasses an integer larger than or equal to1, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 and so forth.

The terms “a” and “an” as used about a nucleotide, an agent, an LNAunit, etc., is intended to mean one or more. In particular, theexpression “a component (such as a nucleotide, an agent, an LNA unit, orthe like) selected from the group consisting of . . . ” is intended tomean that one or more of the cited components may be selected. Thus,expressions like “a component selected from the group consisting of A, Band C” is intended to include all combinations of A, B and C, i.e. A, B,C, A+B, A+C, B+C and A+B+C.

The term “thio-LNA unit” refers to an LNA unit in which X in Scheme 1 isS. A thio-LNA unit can be in both the beta-D form and in the alpha-Lform. Generally, the beta-D form of the thio-LNA unit is preferred. Thebeta-D-form and alpha-L-form of a thio-LNA unit are shown in Scheme 3 ascompounds 3A and 3B, respectively.

The term “amino-LNA unit” refers to an LNA unit in which X in Scheme 1is NH or NR^(H), where R^(H) is hydrogen or C₁₋₄-alkyl. An amino-LNAunit can be in both the beta-D form and in the alpha-L form. Generally,the beta-D form of the amino-LNA unit is preferred. The beta-D-form andalpha-L-form of an amino-LNA unit are shown in Scheme 4 as compounds 4Aand 4B, respectively.

The term “oxy-LNA unit” refers to an LNA unit in which X in Scheme 1 isO. An Oxy-LNA unit can be in both the beta-D form and in the alpha-Lform. Generally, the beta-D form of the oxy-LNA unit is preferred. Thebeta-D form and the alpha-L form of an oxy-LNA unit are shown in Scheme5 as compounds 5A and 5B, respectively.

In the present context, the term “C₁₋₆-alkyl” is intended to mean alinear or branched saturated hydrocarbon chain wherein the longestchains has from one to six carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl,isopentyl, neopentyl and hexyl. A branched hydrocarbon chain is intendedto mean a C₁₋₆-alkyl substituted at any carbon with a hydrocarbon chain.

In the present context, the term “C₁₋₄-alkyl” is intended to mean alinear or branched saturated hydrocarbon chain wherein the longestchains has from one to four carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. Abranched hydrocarbon chain is intended to mean a C₁₋₄-alkyl substitutedat any carbon with a hydrocarbon chain.

When used herein the term “C₁₋₆-alkoxy” is intended to meanC₁₋₆-alkyl-oxy, such as methoxy, ethoxy, n-propoxy, isopropoxy,n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, isopentoxy,neopentoxy and hexoxy.

In the present context, the term “C₂₋₆-alkenyl” is intended to mean alinear or branched hydrocarbon group having from two to six carbon atomsand containing one or more double bonds. Illustrative examples ofC₂₋₆-alkenyl groups include allyl, homo-allyl, vinyl, crotyl, butenyl,butadienyl, pentenyl, pentadienyl, hexenyl and hexadienyl. The positionof the unsaturation (the double bond) may be at any position along thecarbon chain.

In the present context the term “C₂₋₆-alkynyl” is intended to meanlinear or branched hydrocarbon groups containing from two to six carbonatoms and containing one or more triple bonds. Illustrative examples ofC₂₋₆-alkynyl groups include acetylene, propynyl, butynyl, pentynyl andhexynyl. The position of unsaturation (the triple bond) may be at anyposition along the carbon chain. More than one bond may be unsaturatedsuch that the “C₂₋₆-alkynyl” is a di-yne or enedi-yne as is known to theperson skilled in the art.

As used herein, “hybridisation” means hydrogen bonding, which may beWatson-Crick, Hoogsteen, reversed Hoogsteen hydrogen bonding, etc.,between complementary nucleoside or nucleotide bases. The fournucleobases commonly found in DNA are G, A, T and C of which G pairswith C, and A pairs with T. In RNA T is replaced with uracil (U), whichthen pairs with A. The chemical groups in the nucleobases thatparticipate in standard duplex formation constitute the Watson-Crickface. Hoogsteen showed a couple of years later that the purinenucleobases (G and A) in addition to their Watson-Crick face have aHoogsteen face that can be recognised from the outside of a duplex, andused to bind pyrimidine oligonucleotides via hydrogen bonding, therebyforming a triple helix structure.

In the context of the present invention “complementary” refers to thecapacity for precise pairing between two nucleotides sequences with oneanother. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thecorresponding position of a DNA or RNA molecule, then theoligonucleotide and the DNA or RNA are considered to be complementary toeach other at that position. The DNA or RNA strand are consideredcomplementary to each other when a sufficient number of nucleotides inthe oligonucleotide can form hydrogen bonds with correspondingnucleotides in the target DNA or RNA to enable the formation of a stablecomplex. To be stable in vitro or in vivo the sequence of anoligonucleotide need not be 100% complementary to its target microRNA.The terms “complementary” and “specifically hybridisable” thus implythat the oligonucleotide binds sufficiently strong and specific to thetarget molecule to provide the desired interference with the normalfunction of the target whilst leaving the function of non-target RNAsunaffected

In a preferred example the oligonucleotide of the invention is 100%complementary to a human microRNA sequence, such as one of the microRNAsequences referred to herein.

In a preferred example, the oligonucleotide of the invention comprises acontiguous sequence which is 100% complementary to the seed region ofthe human microRNA sequence.

MicroRNAs are short, non-coding RNAs derived from endogenous genes thatact as post-transcriptional regulators of gene expression. They areprocessed from longer (ca 70-80 nt) hairpin-like precursors termedpre-miRNAs by the RNAse III enzyme Dicer. MicroRNAs assemble inribonucleoprotein complexes termed miRNPs and recognize their targetsites by antisense complementarity thereby mediating down-regulation oftheir target genes. Near-perfect or perfect complementarity between themiRNA and its target site results in target mRNA cleavage, whereaslimited complementarity between the microRNA and the target site resultsin translational inhibition of the target gene.

The term “microRNA” or “miRNA”, in the context of the present invention,means an RNA oligonucleotide consisting of between 18 to 25 nucleotides.In functional terms miRNAs are typically regulatory endogenous RNAmolecules.

The terms “target microRNA” or “target miRNA” or “miRNA target” refer toa microRNA with a biological role in human disease, e.g. an upregulated,oncogenic miRNA or a tumor suppressor miRNA in cancer, thereby being atarget for therapeutic intervention of the disease in question.

The terms “target gene” or “target mRNA” refer to regulatory mRNAtargets of microRNAs, in which said “target gene” or “target mRNA” isregulated post-transcriptionally by the microRNA based on near-perfector perfect complementarity between the miRNA and its target siteresulting in target mRNA cleavage; or limited complementarity, oftenconferred to complementarity between the so-called seed sequence(nucleotides 2-7 of the miRNA) and the target site resulting intranslational inhibition of the target mRNA.

In the context of the present invention the oligonucleotide is singlestranded, this refers to the situation where the oligonucleotide is inthe absence of a complementary oligonucleotide—i.e. it is not a doublestranded oligonucleotide complex, such as an siRNA. In one embodiment,the composition according of the invention does not comprise a furtheroligonucleotide which has a region of complementarity with the singlestranded oligonucleotide of five or more consecutive nucleobases, suchas eight or more, or 12 or more of more consecutive nucleobases. It isconsidered that the further oligonucleotide is not covalently linked tothe single stranded oligonucleotide.

LNA-Containing Oligonucleotides of the Invention

While LNA units and non-LNA units may be combined in a number of ways toform oligonucleotides, it has surprisingly been found by the inventorsof the present invention that a certain core DNA sequence and a certainpresence of LNA units in said DNA sequence results in a particularlyhigh inhibition of microRNA. This presence of LNA units in said coresequence of the oligonucleotides of the present invention made saidoligonucleotides highly nuclease resistant.

The nucleotides outside the core sequence may be both LNA units and/ornon-LNA units. In one embodiment, the non-LNA units outside the coresequence are DNA units.

The Core Sequence

In order for the antisense oligonucleotides of the present invention toinhibit their target microRNAs as efficiently as possible there needs tobe a certain degree of complementarity between the antisenseoligonucleotide of the present invention and the corresponding targetmicroRNA.

It is particularly important for the oligonucleotides of the presentinvention to be complementary with positions 3 to 8, counting from the5′ end, of the corresponding target microRNA. Nucleotide 1, countingfrom the 5′ end, in some of the target microRNAs is a non-pairing baseand is most likely hidden in a binding pocket in the Ago 2 protein.Accordingly, the oligonucleotide of the invention may or may not have anucleotide in position 1, counting from the 3′ end, corresponding tonucleotide 1, counting from the 5′ end, of the corresponding targetmicroRNA. In some cases, the first two nucleotides, counting from the 5′end, of the corresponding target microRNA may be left unmatched.

The core sequence of the oligonucleotides of the present invention istherefore a DNA sequence from positions one to six, two to seven orpositions three to eight, counting from the 3′ end, corresponding topositions three to eight, counting from the 5′ end, of the correspondingtarget microRNA.

miR-19b

One particular target microRNA is termed miR-19b. The sequence ofmiR-19b from positions three to eight, counting from the 5′ end, isugcaaa (see GenBank loci AJ421740 and AJ421739). The corresponding DNAsequence is acgttt. The inventors of the present invention havefurthermore found that in order to maximize inhibition of the targetmicroRNAs, the oligonucleotides of the present invention must contain atleast one LNA unit in its core sequence.

Accordingly, a first aspect of the invention relates to anoligonucleotide according to the invention, such as an oligonucleotidehaving a length of from 12 to 26 nucleotides having the DNA sequencefrom positions one to six, two to seven or three to eight, preferablyfrom positions two to seven or three to eight, counting from the 3′ end:

-   -   acgttt,(SEQ ID NO 6)

wherein at least one, such as one, preferably at least two, such as twoor three, DNA units in said sequence have been substituted by theircorresponding LNA unit.

Complementarity with further nucleotides of the target microRNA mayenhance the inhibition of said target microRNA. Therefore, oneembodiment is the oligonucleotide as described above having a DNAsequence from positions one to seven, two to eight or three to nine,preferably from positions two to eight or three to nine, counting fromthe 3′ end:

acgttta, (SEQ ID NO 70)

wherein at least one, such as one, preferably at least two, such as two,more preferably at least three, such as three or four, DNA units in saidsequence have been substituted by their corresponding LNA unit.

In another embodiment, the oligonucleotide according to the presentinvention has a DNA sequence from positions one to eight, two to nine orthree to ten, preferably from positions two to nine or three to ten,counting from the 3′ end:

acgtttag, (SEQ ID NO 71)

wherein at least one, such as one, preferably at least two, such as two,more preferably at least three, such as three or four, DNA units in saidsequence have been substituted by their corresponding LNA unit.

In yet another embodiment, the oligonucleotide according to the presentinvention has a DNA sequence from positions one to nine, two to ten orthree to eleven, preferably from positions two to ten or three toeleven, counting from the 3′ end:

acgtttagg, (SEQ ID NO 72)

wherein at least one, such as one, preferably at least two, such as two,more preferably at least three, such as three, even more preferably atleast four, such as four or five, DNA units in said sequence have beensubstituted by their corresponding LNA unit.

miR-122a

Another interesting target microRNA is miR-122a. The sequence ofmiR-122a from positions three to eight, counting from the 5′ end, isgagugu (see miRBase entry HGNC:MIRN122A). The corresponding DNA sequenceis ctcaca.

Accordingly, a second aspect of the present invention relates to anoligonucleotide according to the invention, such as an oligonucleotidehaving a length of from 12 to 26 nucleotides having the DNA sequencefrom positions one to six, two to seven or three to eight, preferablyfrom positions two to seven or three to eight, counting from the 3′ end:

ctcaca, (SEQ ID NO 7)

wherein at least one, such as one, preferably at least two, such as twoor three, DNA units in said sequence have been substituted by theircorresponding LNA unit.

One embodiment relates to the miR-122a antagomir oligonucleotide asdescribed above having a DNA sequence from positions one to seven, twoto eight or three to nine, preferably from positions two to eight orthree to nine, counting from the 3′ end:

ctcacac,, (SEQ ID NO 73)

wherein at least one, such as one, preferably at least two, such as two,more preferably at least three, such as three or four, DNA units in saidsequence have been substituted by their corresponding LNA unit.

In another embodiment, the oligonucleotide according to the presentinvention has a DNA sequence from positions one to eight, two to nine orthree to ten, preferably from positions two to nine or three to ten,counting from the 3′ end:

ctcacact,, (SEQ ID NO 74)

wherein at least one, such as one, preferably at least two, such as two,more preferably at least three, such as three or four, DNA units in saidsequence have been substituted by their corresponding LNA unit.

In yet another embodiment, the oligonucleotide according to the presentinvention has a DNA sequence from positions one to nine, two to ten orthree to eleven, preferably from positions two to ten or three toeleven, counting from the 3′ end:

ctcacactg,, (SEQ ID NO 75)

wherein at least one, such as one, preferably at least two, such as two,more preferably at least three, such as three, even more preferably atleast four, such as four or five, DNA units in said sequence have beensubstituted by their corresponding LNA unit.

miR-155

Another interesting target microRNA is miR-155. The sequence of miR-155from positions three to eight, counting from the 5′ end, is aaugcu (seemiRBase entry HGNC:MIRN155). The corresponding DNA sequence is ttacga.

Accordingly, a third aspect of the invention relates to anoligonucleotide according to the invention, such as an oligonucleotidehaving a length of from 12 to 26 nucleotides having the DNA sequencefrom positions one to six, two to seven or three to eight, preferablyfrom positions two to seven or three to eight, counting from the 3′ end:

ttacga,, (SEQ ID NO 8)

wherein at least one, such as one, preferably at least two, such as twoor three, DNA units in said sequence have been substituted by theircorresponding LNA unit.

In one embodiment, the miR-155 antagomir oligonucleotide as describedabove has a DNA sequence from positions one to seven, two to eight orthree to nine, preferably from positions two to eight or three to nine,counting from the 3′ end:

ttacgat,, (SEQ ID NO 76)

wherein at least one, such as one, preferably at least two, such as two,more preferably at least three, such as three or four, DNA units in saidsequence have been substituted by their corresponding LNA unit.

In another embodiment, the oligonucleotide according to the presentinvention has a DNA sequence from positions one to eight, two to nine orthree to ten, preferably from positions two to nine or three to ten,counting from the 3′ end:

ttacgatt,, (SEQ ID NO 77)

wherein at least one, such as one, preferably at least two, such as two,more preferably at least three, such as three or four, DNA units in saidsequence have been substituted by their corresponding LNA unit.

In yet another embodiment, the oligonucleotide according to the presentinvention has a DNA sequence from positions one to nine, two to ten orthree to eleven, preferably from positions two to ten or three toeleven, counting from the 3′ end:

ttacgatta,, (SEQ ID NO 78)

wherein at least one, such as one, preferably at least two, such as two,more preferably at least three, such as three, even more preferably atleast four, such as four or five, DNA units in said sequence have beensubstituted by their corresponding LNA unit.

miR-375

Yet another interesting target microRNA is miR-375. The sequence ofmiR-375 from positions three to eight, counting from the 5′ end, isuguucg (see miRBase entry HGNC:MIRN375). The corresponding DNA sequenceis acaagc.

Accordingly, a fourth aspect of the invention relates to anoligonucleotide according to the invention, such as an oligonucleotidehaving a length of from 12 to 26 nucleotides having the DNA sequencefrom positions one to six, two to seven or three to eight, preferablyfrom positions two to seven or three to eight, counting from the 3′ end:

acaagc;, (SEQ ID NO 9)

wherein at least one, such as one, preferably at least two, such as twoor three, DNA units in said sequence have been substituted by theircorresponding LNA unit.

In one embodiment, the miR-375 antagomir oligonucleotide as describeabove has a DNA sequence from positions one to seven, two to eight orthree to nine, preferably from positions two to eight or three to nine,counting from the 3′ end:

acaagca,, (SEQ ID NO 79)

wherein at least one, such as one, preferably at least two, such as two,more preferably at least three, such as three or four, DNA units in saidsequence have been substituted by their corresponding LNA unit.

In another embodiment, the oligonucleotide according to the presentinvention has a DNA sequence from positions one to eight, two to nine orthree to ten, preferably from positions two to nine or three to ten,counting from the 3′ end:

acaagcaa,, (SEQ ID NO 80)

wherein at least one, such as one, preferably at least two, such as two,more preferably at least three, such as three or four, DNA units in saidsequence have been substituted by their corresponding LNA unit.

In yet another embodiment, the oligonucleotide according to the presentinvention has a DNA sequence from positions one to nine, two to ten orthree to eleven, preferably from positions two to ten or three toeleven, counting from the 3′ end:

acaagcaag, (SEQ ID NO 81)

wherein at least one, such as one, preferably at least two, such as two,more preferably at least three, such as three, even more preferably atleast four, such as four or five, DNA units in said sequence have beensubstituted by their corresponding LNA unit.

Modification of Nucleotides in the Core Sequence

As mentioned above, in the core sequence of the oligonucleotides of thepresent invention at least one, such as one, preferably at least two,such as two or three, DNA units in said sequence have been substitutedby their corresponding LNA unit. The present inventors have furtherfound that inhibition of the target microRNAs may be further increasedby making sure that two LNA units in said core sequence are separated byat least one DNA unit.

Accordingly, one embodiment of the invention relates to theoligonucleotide as described above, wherein at least two, such as two orthree, DNA units from positions one to six, two to seven or three toeight, preferably from positions two to seven or three to eight,counting from the 3′ end, have been substituted by their correspondingLNA unit and wherein the LNA units are separated by at least one DNAunit.

The present inventors have also found that inhibition of targetmicroRNAs may be even further increased by making sure that two LNAunits in the core sequence are separated by at most two DNA units.Accordingly, in one embodiment the present relates to theoligonucleotide as described above, wherein the number of consecutiveDNA units from positions one to six, two to seven or three to eight,preferably from positions two to seven or three to eight, counting fromthe 3′ end, is at most two.

Said findings apply to the core sequence per se, i.e. the findingapplies to the positions of the oligonucleotides of the presentinvention corresponding to the core sequence. Hence, another embodimentrelates to the oligonucleotide as described above, wherein at least two,such as two, three or four, DNA units from positions one to seven, twoto eight or three to nine, preferably from positions two to eight orthree to nine, counting from the 3′ end, have been substituted by theircorresponding LNA unit and wherein the LNA units are separated by atleast one DNA unit. A further embodiment relates to the oligonucleotideas described above, wherein the number of consecutive DNA units frompositions one to seven, two to eight or three to nine, preferably frompositions two to eight or three to nine, counting from the 3′ end, is atmost two.

Yet another embodiment relates to the oligonucleotide as describedabove, wherein at least two, such as two, three or four, DNA units frompositions one to eight, two to nine or three to ten, preferably frompositions two to nine or three to ten, counting from the 3′ end, havebeen substituted by their corresponding LNA unit and wherein the LNAunits are separated by at least one DNA unit. Yet a further embodimentrelates to the oligonucleotide as described above, wherein the number ofconsecutive DNA units from positions one to eight, two to nine or threeto ten, preferably from positions two to nine or three to ten, countingfrom the 3′ end, is at most two.

Still another embodiment relates to the oligonucleotide as describedabove, wherein at least two, such as two, three, four or five, DNA unitsfrom positions one to nine, two to ten or three to eleven, preferablyfrom positions two to ten or three to eleven, counting from the 3′ end,have been substituted by their corresponding LNA unit and wherein theLNA units are separated by at least one DNA unit. Still a furtherembodiment relates to the oligonucleotide as described above, whereinthe number of consecutive DNA units from positions one to nine, two toten or three to eleven, preferably from positions two to ten or three toeleven, counting from the 3′ end, is at most two.

Modification of Nucleotides Outside the Core Sequence

As mentioned above, the nucleotides outside the core sequence may beboth LNA units and/or non-LNA units. In one embodiment, the inventionrelates to the oligonucleotide as described above, wherein the number ofLNA units outside the core sequence is at least one, such as one, two,three or four, and wherein said LNA units are separated by at least onenon-LNA unit. In a further embodiment, the substitution pattern outsidethe core sequence is such that the number of consecutive non-LNA unitsoutside the core sequence is at most two.

Modification of Nucleotides in Positions 3 to 8, Counting from the 3′End

In the following embodiments which refer to the modification ofnucleotides in positions 3 to 8, counting from the 3′ end, the LNA unitsmay be replaced with other nucleotide analogues, such as those referredto herein. “X” may, therefore be selected from the group consisting of2′-O-alkyl-RNA unit, 2′-OMe-RNA unit, 2′-amino-DNA unit, 2′-fluoro-DNAunit, LNA unit, PNA unit, HNA unit, INA unit. “x” is preferably DNA orRNA, most preferably DNA.

In an interesting embodiment of the invention, the oligonucleotides ofthe invention are modified in positions 3 to 8, counting from the 3′end. The design of this sequence may be defined by the number of non-LNAunits present or by the number of LNA units present. In a preferredembodiment of the former, at least one, such as one, of the nucleotidesin positions three to eight, counting from the 3′ end, is a non-LNAunit. In another embodiment, at least two, such as two, of thenucleotides in positions three to eight, counting from the 3′ end, arenon-LNA units. In yet another embodiment, at least three, such as three,of the nucleotides in positions three to eight, counting from the 3′end, are non-LNA units. In still another embodiment, at least four, suchas four, of the nucleotides in positions three to eight, counting fromthe 3′ end, are non-LNA units. In a further embodiment, at least five,such as five, of the nucleotides in positions three to eight, countingfrom the 3′ end, are non-LNA units. In yet a further embodiment, all sixnucleotides in positions three to eight, counting from the 3′ end, arenon-LNA units. In a preferred embodiment, said non-LNA unit is a DNAunit.

Alternatively defined, in a preferred embodiment, the oligonucleotideaccording to the invention comprises at least one LNA unit in positionsthree to eight, counting from the 3′ end. In an embodiment thereof, theoligonucleotide according to the present invention comprises one LNAunit in positions three to eight, counting from the 3′ end. Thesubstitution pattern for the nucleotides in positions three to eight,counting from the 3′ end, may be selected from the group consisting ofXxxxxx, xXxxxx, xxXxxx, xxxXxx, xxxxXx and xxxxxX, wherein “X” denotesan LNA unit and “x” denotes a non-LNA unit.

In another embodiment, the oligonucleotide according to the presentinvention comprises at least two LNA units in positions three to eight,counting from the 3′ end. In an embodiment thereof, the oligonucleotideaccording to the present invention comprises two LNA units in positionsthree to eight, counting from the 3′ end. The substitution pattern forthe nucleotides in positions three to eight, counting from the 3′ end,may be selected from the group consisting of XXxxxx, XxXxxx, XxxXxx,XxxxXx, XxxxxX, xXXxxx, xXxXxx, xXxxXx, xXxxxX, xxXXxx, xxXxXx, xxXxxX,xxxXXx, xxxXxX and xxxxXX, wherein “X” denotes an LNA unit and “x”denotes a non-LNA unit. In a preferred embodiment, the substitutionpattern for the nucleotides in positions three to eight, counting fromthe 3′ end, is selected from the group consisting of XxXxxx, XxxXxx,XxxxXx, XxxxxX, xXxXxx, xXxxXx, xXxxxX, xxXxXx, xxXxxX and xxxXxX,wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit. In amore preferred embodiment, the substitution pattern for the nucleotidesin positions three to eight, counting from the 3′ end, is selected fromthe group consisting of xXxXxx, xXxxXx, xXxxxX, xxXxXx, xxXxxX andxxxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.In an even more preferred embodiment, the substitution pattern for thenucleotides in positions three to eight, counting from the 3′ end, isselected from the group consisting of xXxXxx, xXxxXx and xxXxXx, wherein“X” denotes an LNA unit and “x” denotes a non-LNA unit. In a mostpreferred embodiment, the substitution pattern for the nucleotides inpositions three to eight, counting from the 3′ end, is xXxXxx, wherein“X” denotes an LNA unit and “x” denotes a non-LNA unit.

In yet another embodiment, the oligonucleotide according to the presentinvention comprises at least three LNA units in positions three toeight, counting from the 3′ end. In an embodiment thereof, theoligonucleotide according to the present invention comprises three LNAunits in positions three to eight, counting from the 3′ end. Thesubstitution pattern for the nucleotides in positions three to eight,counting from the 3′ end, may be selected from the group consisting ofXXXxxx, xXXXxx, xxXXXx, xxxXXX, XXxXxx, XXxxXx, XXxxxX, xXXxXx, xXXxxX,xxXXxX, XxXXxx, XxxXXx, XxxxXX, xXxXXx, xXxxXX, xxXxXX, xXxXxX andXxXxXx, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.In a preferred embodiment, the substitution pattern for the nucleotidesin positions three to eight, counting from the 3′ end, is selected fromthe group consisting of XXxXxx, XXxxXx, XXxxxX, xXXxXx, xXXxxX, xxXXxX,XxXXxx, XxxXXx, XxxxXX, xXxXXx, xXxxXX, xxXxXX, xXxXxX and XxXxXx,wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit. In amore preferred embodiment, the substitution pattern for the nucleotidesin positions three to eight, counting from the 3′ end, is selected fromthe group consisting of xXXxXx, xXXxxX, xxXXxX, xXxXXx, xXxxXX, xxXxXXand xXxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNAunit. In an even more preferred embodiment, the substitution pattern forthe nucleotides in positions three to eight, counting from the 3′ end,is xXxXxX or XxXxXx, wherein “X” denotes an LNA unit and “x” denotes anon-LNA unit. In a most preferred embodiment, the substitution patternfor the nucleotides, in positions three to eight, counting from the 3′end, is xXxXxX, wherein “X” denotes an LNA unit and “x” denotes anon-LNA unit.

In a further embodiment, the oligonucleotide according to the presentinvention comprises at least four LNA units in positions three to eight,counting from the 3′ end. In an embodiment thereof, the oligonucleotideaccording to the present invention comprises four LNA units in positionsthree to eight, counting from the 3′ end. The substitution pattern forthe nucleotides in positions three to eight, counting from the 3′ end,may be selected from the group consisting of xxXXXX, xXxXXX, xXXxXX,xXXXxX, xXXXXx, XxxXXX, XxXxXX, XxXXxX, XxXXXx, XXxxXX, XXxXxX, XXxXXx,XXXxxX, XXXxXx and XXXXxx, wherein “X” denotes an LNA unit and “x”denotes a non-LNA unit.

In yet a further embodiment, the oligonucleotide according to thepresent invention comprises at least five LNA units in positions threeto eight, counting from the 3′ end. In an embodiment thereof, theoligonucleotide according to the present invention comprises five LNAunits in positions three to eight, counting from the 3′ end. Thesubstitution pattern for the nucleotides in positions three to eight,counting from the 3′ end, may be selected from the group consisting ofxXXXXX, XxXXXX, XXxXXX, XXXxXX, XXXXxX and XXXXXx, wherein “X” denotesan LNA unit and “x” denotes a non-LNA unit.

Preferably, the oligonucleotide according to the present inventioncomprises one or two LNA units in positions three to eight, countingfrom the 3′ end. This is considered advantageous for the stability ofthe A-helix formed by the oligo:microRNA duplex, a duplex resembling anRNA: RNA duplex in structure.

In a preferred embodiment, said non-LNA unit is a DNA unit.

Variation of the Length of the Oligonucleotides

The length of the oligonucleotides of the invention need not match thelength of the target microRNAs exactly. Indeed it is consideredadvantageous to have short oligonucleotides, such as between 10-17 or10-16 nucleobases.

In one embodiment, the oligonucleotide according to the present has alength of from 8 to 24 nucleotides, such as 10 to 24, between 12 to 24nucleotides, such as a length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23 or 24 nucleotides, preferably a length of from10-22, such as between 12 to 22 nucleotides, such as a length of 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 nucleotides, morepreferably a length of from 10-20, such as between 12 to 20 nucleotides,such as a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20nucleotides, even more preferably a length of from 10 to 19, such asbetween 12 to 19 nucleotides, such as a length of 10, 11, 12, 13, 14,15, 16, 17, 18 or 19 nucleotides, e.g. a length of from 10 to 18, suchas between 12 to 18 nucleotides, such as a length of 10, 11, 12, 13, 14,15, 16, 17 or 18 nucleotides, more preferably a length of from 10-17,such as from 12 to 17 nucleotides, such as a length of 10, 11, 12, 13,14, 15, 16 or 17 nucleotides, most preferably a length of from 10 to 16,such as between 12 to 16 nucleotides, such as a length of 10, 11, 12,13, 14, 15 or 16 nucleotides.

Modification of nucleotides from position 11, counting from the 3′ end,to the 5′ end

The substitution pattern for the nucleotides from position 11, countingfrom the 3′ end, to the 5′ end may include nucleotide analogue units(such as LNA) or it may not. In a preferred embodiment, theoligonucleotide according to the present invention comprises at leastone nucleotide analogue unit (such as LNA), such as one nucleotideanalogue unit, from position 11, counting from the 3′ end, to the 5′end. In another preferred embodiment, the oligonucleotide according tothe present invention comprises at least two nucleotide analogue units,such as LNA units, such as two nucleotide analogue units, from position11, counting from the 3′ end, to the 5′ end.

In the following embodiments which refer to the modification ofnucleotides in the nucleobases from position 11 to the 5′ end of theoligonucleotide, the LNA units may be replaced with other nucleotideanalogues, such as those referred to herein. “X” may, therefore beselected from the group consisting of 2′-O-alkyl-RNA unit, 2′-OMe-RNAunit, 2′-amino-DNA unit, 2′-fluoro-DNA unit, LNA unit, PNA unit, HNAunit, INA unit. “x” is preferably DNA or RNA, most preferably DNA.

In one embodiment, the oligonucleotide according to the presentinvention has the following substitution pattern, which is repeated fromnucleotide eleven, counting from the 3′ end, to the 5′ end: xXxX orXxXx, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit. Inanother embodiment, the oligonucleotide according to the presentinvention has the following substitution pattern, which is repeated fromnucleotide eleven, counting from the 3′ end, to the 5′ end: XxxXxx,xXxxXx or xxXxxX, wherein “X” denotes an LNA unit and “x” denotes anon-LNA unit. In yet another embodiment, the oligonucleotide accordingto the present invention has the following substitution pattern, whichis repeated from nucleotide eleven, counting from the 3′ end, to the 5′end: XxxxXxxx, xXxxxXxx, xxXxxxXx or xxxXxxxX, wherein “X” denotes anLNA unit and “x” denotes a non-LNA unit.

The specific substitution pattern for the nucleotides from position 11,counting from the 3′ end, to the 5′ end depends on the number ofnucleotides in the oligonucleotides according to the present invention.In a preferred embodiment, the oligonucleotide according to the presentinvention contains 12 nucleotides and the substitution pattern forpositions 11 to 12, counting from the 3′ end, is selected from the groupconsisting of xX and Xx, wherein “X” denotes an LNA unit and “x” denotesa non-LNA unit. In a more preferred embodiment thereof, the substitutionpattern for positions 11 to 12, counting from the 3′ end, is xX, wherein“X” denotes an LNA unit and “x” denotes a non-LNA unit. Alternatively,no LNA units are present in positions 11 to 12, counting from the 3′end, i.e. the substitution pattern is xx.

In another preferred embodiment, the oligonucleotide according to thepresent invention contains 13 nucleotides and the substitution patternfor positions 11 to 13, counting from the 3′ end, is selected from thegroup consisting of Xxx, xXx, xxX, XXx, XxX, xXX and XXX, wherein “X”denotes an LNA unit and “x” denotes a non-LNA unit. In a more preferredembodiment thereof, the substitution pattern for positions 11 to 13,counting from the 3′ end, is selected from the group consisting of xXx,xxX and xXX, wherein “X” denotes an LNA unit and “x” denotes a non-LNAunit. In a most preferred embodiment thereof, the substitution patternfor positions 11 to 13, counting from the 3′ end, is xxX, wherein “X”denotes an LNA unit and “x” denotes a non-LNA unit. Alternatively, noLNA units are present in positions 11 to 13, counting from the 3′ end,i.e. the substitution pattern is xxx.

In yet another preferred embodiment, the oligonucleotide according tothe present invention contains 14 nucleotides and the substitutionpattern for positions 11 to 14, counting from the 3′ end, is selectedfrom the group consisting of Xxxx, xXxx, xxXx, xxxX, XXxx, XxXx, XxxX,xXXx, xXxX and xxXX, wherein “X” denotes an LNA unit and “x” denotes anon-LNA unit. In a preferred embodiment thereof, the substitutionpattern for positions 11 to 14, counting from the 3′ end, is selectedfrom the group consisting of xXxx, xxXx, xxxX, xXxX and xxXX, wherein“X” denotes an LNA unit and “x” denotes a non-LNA unit. In a morepreferred embodiment thereof, the substitution pattern for positions 11to 14, counting from the 3′ end, is xXxX, wherein “X” denotes an LNAunit and “x” denotes a non-LNA unit. Alternatively, no LNA units arepresent in positions 11 to 14, counting from the 3′ end, i.e. thesubstitution pattern is xxxx

In a further preferred embodiment, the oligonucleotide according to thepresent invention contains 15 nucleotides and the substitution patternfor positions 11 to 15, counting from the 3′ end, is selected from thegroup consisting of Xxxxx, xXxxx, xxXxx, xxxXx, xxxxX, XXxxx, XxXxx,XxxXx, XxxxX, xXXxx, xXxXx, xXxxX, xxXXx, xxXxX, xxxXX and XxXxX,wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit. In apreferred embodiment thereof, the substitution pattern for positions 11to 15, counting from the 3′ end, is selected from the group consistingof xxXxx, XxXxx, XxxXx, xXxXx, xXxxX and xxXxX, wherein “X” denotes anLNA unit and “x” denotes a non-LNA unit. In a more preferred embodimentthereof, the substitution pattern for positions 11 to 15, counting fromthe 3′ end, is selected from the group consisting of xxXxx, xXxXx, xXxxXand xxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNAunit. In an even more preferred embodiment thereof, the substitutionpattern for positions 11 to 15, counting from the 3′ end, is selectedfrom the group consisting of xXxxX and xxXxX, wherein “X” denotes an LNAunit and “x” denotes a non-LNA unit. In a most preferred embodiment, thesubstitution pattern for positions 11 to 15, counting from the 3′ end,is xxXxX, wherein “X” denotes an LNA unit and “x” denotes a non-LNAunit. Alternatively, no LNA units are present in positions 11 to 15,counting from the 3′ end, i.e. the substitution pattern is xxxxx

In yet a further preferred embodiment, the oligonucleotide according tothe present invention contains 16 nucleotides and the substitutionpattern for positions 11 to 16, counting from the 3′ end, is selectedfrom the group consisting of Xxxxxx, xXxxxx, xxXxxx, xxxXxx, xxxxXx,xxxxxX, XXxxxx, XxXxxx, XxxXxx, XxxxXx, XxxxxX, xXXxxx, xXxXxx, xXxxXx,xXxxxX, xxXXxx, xxXxXx, xxXxxX, xxxXXx, xxxXxX, xxxxXX, XXXxxx, XXxXxx,XXxxXx, XXxxxX, XxXXxx, XxXxXx, XxXxxX, XxxXXx, XxxXxX, XxxxXX, xXXXxx,xXXxXx, xXXxxX, xXxXXx, xXxXxX, xXxxXX, xxXXXx, xxXXxX, xxXxXX andxxxXXX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.In a preferred embodiment thereof, the substitution pattern forpositions 11 to 16, counting from the 3′ end, is selected from the groupconsisting of XxxXxx, xXxXxx, xXxxXx, xxXxXx, xxXxxX, XxXxXx, XxXxxX,XxxXxX, xXxXxX, xXxxXX and xxXxXX, wherein “X” denotes an LNA unit and“x” denotes a non-LNA unit. In a more preferred embodiment thereof, thesubstitution pattern for positions 11 to 16, counting from the 3′ end,is selected from the group consisting of xXxXxx, xXxxXx, xxXxXx, xxXxxX,xXxXxX, xXxxXX and xxXxXX, wherein “X” denotes an LNA unit and “x”denotes a non-LNA unit. In an even more preferred embodiment thereof,the substitution pattern for positions 11 to 16, counting from the 3′end, is selected from the group consisting of xxXxxX, xXxXxX, xXxxXX andxxXxXX, wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit.In a still more preferred embodiment thereof, the substitution patternfor positions 11 to 16, counting from the 3′ end, is selected from thegroup consisting of xxXxxX and xXxXxX, wherein “X” denotes an LNA unitand “x” denotes a non-LNA unit. In a most preferred embodiment thereof,the substitution pattern for positions 11 to 16, counting from the 3′end, is xxXxxX, wherein “X” denotes an LNA unit and “x” denotes anon-LNA unit. Alternatively, no LNA units are present in positions 11 to16, counting from the 3′ end, i.e. the substitution pattern is xxxxxx

In a preferred embodiment of the invention, the oligonucleotideaccording to the present invention contains an LNA unit at the 5′ end.In another preferred embodiment, the oligonucleotide according to thepresent invention contains an LNA unit at the first two positions,counting from the 5′ end.

In a particularly preferred embodiment, the oligonucleotide according tothe present invention contains 13 nucleotides and the substitutionpattern, starting from the 3′ end, is XXxXxXxxXXxxX, wherein “X” denotesan LNA unit and “x” denotes a non-LNA unit. The preferred sequence forthis embodiment, starting from the 3′ end, is CCtCaCacTGttA, wherein acapital letter denotes a nitrogenous base in an LNA-unit and a smallletter denotes a nitrogenous base in a non-LNA unit.

In another particularly preferred embodiment, the oligonucleotideaccording to the present invention contains 15 nucleotides and thesubstitution pattern, starting from the 3′ end, is XXxXxXxxXXxxXxX,wherein “X” denotes an LNA unit and “x” denotes a non-LNA unit. Thepreferred sequence for this embodiment, starting from the 3′ end, isCCtCaCacTGttAcC, wherein a capital letter denotes a nitrogenous base inan LNA-unit and a small letter denotes a nitrogenous base in a non-LNAunit.

Modification of the Internucleoside Linkage Group

Typical internucleoside linkage groups in oligonucleotides are phosphategroups, but these may be replaced by internucleoside linkage groupsdiffering from phosphate. In a further interesting embodiment of theinvention, the oligonucleotide of the invention is modified in itsinternucleoside linkage group structure, i.e. the modifiedoligonucleotide comprises an internucleoside linkage group which differsfrom phosphate. Accordingly, in a preferred embodiment, theoligonucleotide according to the present invention comprises at leastone

Specific examples of internucleoside linkage groups which differ fromphosphate

(—O—P(O)₂—O—) include —O—P(O,S)—O—, —O—P(S)₂—O—, —S—P(O)₂—O—,—S—P(O,S)—O—, —S—P(S)₂—O—, —O—P(O)₂—S—, —O—P(O,S)—S—, —S—P(O)₂—S—,—O—PO(R^(H))—O—, O—PO(OCH₃)—O—, —O—PO(NR^(H))—O—, —O—PO(OCH₂CH₂S—R)—O—,—O—PO(BH₃)—O—, —O—PO(NHR^(H))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—,—NR^(H)—CO—O—, —NR^(H)—CO—NR^(H)—, —O—CO—O—, —O—CO—NR^(H)—,—NR^(H)—CO—CH₂—, —O—CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—, —CO—NR^(H)—CH₂—,—CH₂—NR^(H)—CO—, —O—CH₂—CH₂—S—, —S—CH₂—CH₂—O—, —S—CH₂—CH₂—S—,—CH₂—SO₂—CH₂—, —CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—CO—, —CH₂—NCH₃—O—CH₂—,where R^(H) is hydrogen or C₁₋₄-alkyl.

When the internucleoside linkage group is modified, the internucleosidelinkage group is preferably a phosphorothioate group (—O—P(O,S)—O—). Ina preferred embodiment, all internucleoside linkage groups of theoligonucleotides according to the present invention arephosphorothioate.

Designs for Specific microRNAs

The following table provides examples of oligonucleotide according tothe present invention, such as those used in pharmaceuticalcompositions, as compared to prior art type of molecules.

Oligo #, target microRNA, oligo sequence Design SEQ ID target:hsa-miR-122a MIMAT0000421 uggagugugacaaugguguuugu SEQ ID NO 1 screenedin HUH-7 cell line expressing miR-122 3962: miR-1225′-ACAAacaccattgtcacacTCCA-3′ Full complement, gap SEQ ID NO 11 3965:miR-122 5′-acaaacACCATTGTcacactcca-3′ Full complement, block SEQ ID NO12 3972: miR-122 5′-acAaaCacCatTgtCacActCca-3′ Full complement, LNA_3SEQ ID NO 13 3549 (3649): miR-122 5′-CcAttGTcaCaCtCC-3′ New design SEQID NO 14 3975: miR-122 5′-CcAtTGTcaCACtCC-3′ Enhanced new design SEQ IDNO 15 3975′: miR-122 5′-ATTGTcACACtCC-3′ ED-13mer SEQ ID NO 16 3975″:m1R-122 5′-TGTcACACtCC-3′ ED-11mer SEQ ID NO 17 3549′ (3649): miR-1225′ CC^(M)AT^(M)T^(M)GTC^(M)A^(M)CA^(M)CT^(M)CC-3′ New design-2′MOE SEQID NO 18 3549″ (3649): miR-1225′ CC^(F)AT^(F)T^(F)GTC^(F)A^(F)CA^(F)CT^(F)CC-3′ New design-2′FluoroSEQ ID NO 19 target: hsa-miR-19b MIMAT0000074 ugugcaaauccaugcaaaacugaSEQ ID NO 2 screened HeLa cell line expressing miR-19b 3963: miR-19b5′-TCAGttttgcatggatttgCACA-3′ Full complement, gap SEQ ID NO 20 3967:miR-19b 5′-tcagttTTGCATGGatttgcaca-3′ Full complement, block SEQ ID NO21 3973: miR-19b 5′-tcAgtTttGcaTggAttTgcAca-3′ Full complement, LNA_3SEQ ID NO 22 3560: miR-19b 5′-TgCatGGatTtGcAC-3′ New design SEQ ID NO 233976: miR-19b 5′-TgCaTGGatTTGcAC-3′ Enhanced new design SEQ ID NO 243976′: miR-19b 5′-CaTGGaTTTGcAC-3′ ED-13mer SEQ ID NO 25 3976″: miR-19b5′-TGGaTTTGcAC-3′ ED-11mer SEQ ID NO 26 3560′: miR-19b5′ TG^(M)CA^(M)T^(M)GGA^(M)T^(M)TT^(M)GC^(M)AC-3′ New design-2′MOE SEQID NO 27 3560″: miR-19b5′-TG^(F)CA^(F)T^(F)GGA^(F)T^(F)TT^(F)GC^(F)AC-3′ New design-2′MOE SEQID NO 28 target: hsa-miR-155 MIMAT0000646 uuaaugcuaaucgugauagggg SEQ IDNO 3 screen in 518A2 cell line expressing miR-155 3964: miR-1555′-CCCCtatcacgattagcaTTAA-3′ Full complement, gap SEQ ID NO 29 3968:miR-155 5′-cccctaTCACGATTagcattaa-3′ Full complement, block SEQ ID NO 303974: miR-155 5′-cCccTatCacGatTagCatTaa-3′ Full complement, LNA_3 SEQ IDNO 31 3758: miR-155 5′-TcAcgATtaGcAtTA-3′ New design SEQ ID NO 32 3818:miR-155 5′-TcAcGATtaGCAtTA-3′ Enhanced new design SEQ ID NO 33 3818′:miR-155 5′-ACGATtAGCAtTA-3′ ED-13mer SEQ ID NO 34 3818″: miR-1555′-GATtAGCaTTA-3′ ED-11mer SEQ ID NO 35 3758′: miR-1555′-TC^(M)AC^(M)G^(M)ATTA^(M)GC^(M)AT^(M)TA-3′ New design-2′MOE SEQ ID NO36 3758″: miR-155 5′-TC^(F)AC^(F)G^(F)ATT^(F)A^(F)GC^(F)AT^(F)TA-3′ Newdesign-2′Fluoro SEQ ID NO 37 target: hsa-miR-21 MIMAT0000076uagcuuaucagacugauguuga SEQ ID NO 4 miR-21 5′-TCAAcatcagtctgataaGCTA-3′Full complement, gap SEQ ID NO 38 miR-21 5′-tcaacaTCAGTCTGataagcta-3′Full complement, block SEQ ID NO 39 miR-21 5′-tcAtcAtcAgtCtgAtaAGcTt-3′Full complement, LNA_3 SEQ ID NO 40 miR-21 5′-TcAgtCTgaTaAgCT-3′ Newdesign SEQ ID NO 41 miR-21 5′-TcAgTCTgaTAAgCT-3′- Enhanced new designSEQ ID NO 42 miR-21 5′-AGTCTgATAAgCT-3′- ED-13mer SEQ ID NO 43 miR-215′-TCTgAtAAGCT-3′- ED-11mer SEQ ID NO 44 miR-215′-TC^(M)AG^(M)T^(M)CTG^(M)A^(M)TA^(M)AG^(M)CT-3′ New design-2′MOE SEQID NO 45 miR-21 5′-TC^(F)AG^(F)T^(F)CTG^(F)A^(F)TA^(F)AG^(F)CT-3′ Newdesign-2′Fluoro SEQ ID NO 46 target: hsa-miR-375 MIMAT0000728uuuguucguucggcucgcguga SEQ ID NO 5 miR-375 5′-TCTCgcgtgccgttcgttCTTT-3′Full complement, gap SEQ ID NO 47 miR-375 5′-tctcgcGTGCCGTTcgttcttt-3′Full complement, block SEQ ID NO 48 miR-375 5′-tcTcgCgtGccGttCgtTctTt-3′Full complement, LNA_3 SEQ ID NO 49 miR-375 5′-GtGccGTtcGtTcTT 3′ Newdesign SEQ ID NO 50 miR-375 5′-GtGcCGTtcGTTcTT 3′ Enhanced new designSEQ ID NO 51 miR-375 5′-GCCGTtCgTTCTT 3′ ED-13mer SEQ ID NO 52 miR-3755′-CGTTcGTTCTT 3′ ED-11mer SEQ ID NO 53 miR-3755′-GT^(M)GC^(M)C^(M)GTT^(M)C^(M)GT^(M)TC^(M)TT 3′ New design-2′MOE SEQID NO 54 miR-375 5′-GT^(F)GC^(F)C^(F)GTT^(F)C^(F)GT^(F)TC^(F)TT 3′ Newdesign-2′Fluoro SEQ ID NO 55

Capital Letters without a superscript M or F, refer to LNA units. Lowercase=DNA, except for lower case in bold=RNA. The LNA cytosines mayoptionally be methylated). Capital letters followed by a superscript Mrefer to 2′OME RNA units, Capital letters followed by a superscript Frefer to 2′fluoro DNA units, lowercase letter refer to DNA. The aboveoligos may in one embodiment be entirely phosphorothioate, but othernucleobase linkages as herein described bay be used. In one embodimentthe nucleobase linkages are all phosphodiester. It is considered thatfor use within the brain/spinal cord it is preferable to usephosphodiester linkages, for example for the use of antimiRs targetingmiR21.

The oligonucleotides according to the invention may, in one embodiment,have a sequence of nucleobases 5′-3′ selected form the group consistingof:

LdLddLLddLdLdLL (New design) LdLdLLLddLLLdLL (Enhanced new design)LMLMMLLMMLMLMLL (New design-2′MOE) LMLMLLLMMLLLMLL (Enhanced newdesign-2′MOE) LFLFFLLFFLFLFLL (New design-2′ Fluoro) LFLFLLLFFLLLFLL(Enhanced new design-2′ Fluoro) LddLddLddL(d)(d)(L)(d)(d)(L)(d) ‘Everythird’ dLddLddLdd(L)(d)(d)(L)(d)(d)(L) ‘Every third’ddLddLddLd(d)(L)(d)(d)(L)(d)(d) ‘Every third’LMMLMMLMML(M)(M)(L)(M)(M)(L)(M) ‘Every third’MLMMLMMLMM(L)(M)(M)(L)(M)(M)(L) ‘Every third’MMLMMLMMLM(M)(L)(M)(M)(L)(M)(M) ‘Every third’LFFLFFLFFL(F)(F)(L)(F)(F)(L)(F) ‘Every third’FLFFLFFLFF(L)(F)(F)(L)(F)(F)(L) ‘Every third’FFLFFLFFLF(F)(L)(F)(F)(L)(F)(F) ‘Every third’dLdLdLdLdL(d)(L)(d)(L)(d)(L)(d) ‘Every second’LdLdLdLdL(d)(L)(d)(L)(d)(L)(d)(L) ‘Every second’MLMLMLMLML(M)(L)(M)(L)(M)(L)(M) ‘Every second’LMLMLMLML(M)(L)(M)(L)(M)(L)(M)(L) ‘Every second’FLFLFLFLFL(F)(L)(F)(L)(F)(L)(F) ‘Every second’LFLFLFLFL(F)(L)(F)(L)(F)(L)(F)(L) ‘Every second’

Wherein L=LNA unit, d=DNA units, M=2′MOE RNA, F=2′Fluoro and residues inbrackets are optional

Specific examples of the oligonucleotides according to the presentinvention may be selected from the group consisting of tgCatGgaTttGcaCa(SEQ ID NO 82), tgCatGgaTttGcaC (SEQ ID NO 83), CatGgaTttGcaC (SEQ ID NO84), tGcAtGgAtTtGcAc (SEQ ID NO 85), cAtGgAtTtGcAc (SEQ ID NO 86),CatGGatTtGcAC (SEQ ID NO 87), TgCatGGatTtGcAC (SEQ ID NO 88),TgCaTgGaTTtGcACa (SEQ ID NO 89), cCatTgtCacActCca (SEQ ID NO 90),cCatTgtAacTctCca (SEQ ID NO 91), ccAttGtcAcaCtcCa (SEQ ID NO 92),cCatTgtCacActCc (SEQ ID NO 93), atTgtCacActCc (SEQ ID NO 94),ccAttGtcAcaCtcC (SEQ ID NO 95), AttGtcAcaCtcC (SEQ ID NO 96),aTtGtCaCaCtCc (SEQ ID NO 97), AttGTcaCaCtCC (SEQ ID NO 98),CcAttGTcaCaCtCC (SEQ ID NO 99), CcaTtgTcacActcCa (SEQ ID NO 100),CCAttgtcacacTCCa (SEQ ID NO 101), tCacGatTagCatTaa (SEQ ID NO 102),aTcaCgaTtaGcaTta (SEQ ID NO 103), TcAcGaTtAgCaTtAa (SEQ ID NO 104),AtcAcGaTtAgCaTta (SEQ ID NO 105), gAgcCgaAcgAacAa (SEQ ID NO 106),gcCgaAcgAacAa (SEQ ID NO 107), GaGcCgAaCgAaCaA (SEQ ID NO 108), andGcCgAaCgAaCaA (SEQ ID NO 109); wherein a lowercase letter identifies thenitrogenous base of a DNA unit and an uppercase letter identifies thenitrogenous base of an LNA unit, with uppercase C referring to ^(Me)C.

It will be recognised that the design of LNA/DNA nucleobases in theabove specific examples may be applied to other oligonucleotidesaccording to the invention.

Conjugates

The invention also provides for conjugates comprising theoligonucleotide according to the invention.

In one embodiment of the invention the oligomeric compound is linked toligands/conjugates, which may be used, e.g. to increase the cellularuptake of antisense oligonucleotides. This conjugation can take place atthe terminal positions 5′/3′-OH but the ligands may also take place atthe sugars and/or the bases. In particular, the growth factor to whichthe antisense oligonucleotide may be conjugated, may comprisetransferrin or folate. Transferrin-polylysine-oligonucleotide complexesor folate-polylysine-oligonucleotide complexes may be prepared foruptake by cells expressing high levels of transferrin or folatereceptor. Other examples of conjugates/ligands are cholesterol moieties,duplex intercalators such as acridine, poly-L-lysine, “end-capping” withone or more nuclease-resistant linkage groups such asphosphoromonothioate, and the like. The invention also provides for aconjugate comprising the compound according to the invention as hereindescribed, and at least one non-nucleotide or non-polynucleotide moietycovalently attached to said compound. Therefore, in one embodiment wherethe compound of the invention consists of s specified nucleic acid, asherein disclosed, the compound may also comprise at least onenon-nucleotide or non-polynucleotide moiety (e.g. not comprising one ormore nucleotides or nucleotide analogues) covalently attached to saidcompound. The non-nucleobase moiety may for instance be or comprise asterol such as cholesterol.

Therefore, it will be recognised that the oligonucleotide of theinvention, such as the oligonucleotide used in pharmaceutical(therapeutic) formulations may comprise further non-nucleobasecomponents, such as the conjugates herein defined.

The LNA Unit

In a preferred embodiment, the LNA unit has the general chemicalstructure shown in Scheme 1 below:

wherein

-   -   X is selected from the group consisting of O, S and NR^(H),        where R^(H) is H or C₁₋₄-alkyl;    -   Y is (—CH₂)_(r), where r is an integer of 1-4; and    -   B is a nitrogenous base.

In a preferred embodiment of the invention, r is 1or 2, in particular 1,i.e. a preferred LNA unit has the chemical structure shown in Scheme 2below:

wherein X and B are as defined above.

In an interesting embodiment, the LNA units incorporated in theoligonucleotides of the invention are independently selected from thegroup consisting of thio-LNA units, amino-LNA units and oxy-LNA units.

Thus, the thio-LNA unit may have the chemical structure shown in Scheme3 below:

wherein B is as defined above.

Preferably, the thio-LNA unit is in its beta-D-form, i.e. having thestructure shown in 3A above.

likewise, the amino-LNA unit may have the chemical structure shown inScheme 4 below:

wherein B and R^(H) are as defined above.

Preferably, the amino-LNA unit is in its beta-D-form, i.e. having thestructure shown in 4A above.

The oxy-LNA unit may have the chemical structure shown in Scheme 5below:

wherein B is as defined above.

Preferably, the oxy-LNA unit is in its beta-D-form, i.e. having thestructure shown in 5A above.

As indicated above, B is a nitrogenous base which may be of natural ornon-natural origin. Specific examples of nitrogenous bases includeadenine (A), cytosine (C), 5-methylcytosine (^(Me)C), isocytosine,pseudoisocytosine, guanine (G), thymine (T), uracil (U), 5-bromouracil,5-propynyluracil, 5-propyny-6, 5-methyithiazoleuracil, 6-aminopurine,2-aminopurine, inosine, 2,6-diaminopurine, 7-propyne-7-deazaadenine,7-propyne-7-deazaguanine and 2-chloro-6-aminopurine.

Terminal Groups

Specific examples of terminal groups include terminal groups selectedfrom the group consisting of hydrogen, azido, halogen, cyano, nitro,hydroxy, Prot-O—, Act-O—, mercapto, Prot-S—, Act-S—, C₁₋₆-alkylthio,amino, Prot-N(R^(H))—, Act-N(R^(H))—, mono- or di(C₁₋₆-alkyl)amino,optionally substituted C₁₋₆-alkoxy, optionally substituted C₁₋₆-alkyl,optionally substituted C₂₋₆-alkenyl, optionally substitutedC₂₋₆-alkenyloxy, optionally substituted C₂₋₆-alkynyl, optionallysubstituted C₂₋₆-alkynyloxy, monophosphate including protectedmonophosphate, monothiophosphate including protected monothiophosphate,diphosphate including protected diphosphate, dithiophosphate includingprotected dithiophosphate, triphosphate including protectedtriphosphate, trithiophosphate including protected trithiophosphate,where Prot is a protection group for —OH, —SH and —NH(R^(H)), and Act isan activation group for —OH, —SH, and —NH(R^(H)), and R^(H) is hydrogenor C₁₋₆-alkyl.

Examples of phosphate protection groups include S-acetylthioethyl (SATE)and S-pivaloylthioethyl (t-butyl-SATE).

Still further examples of terminal groups include DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, ligands, carboxy, sulphono, hydroxymethyl,Prot-O—CH₂—, Act-O—CH₂—, aminomethyl, Prot-N(R^(H))—CH₂—,Act-N(R^(H))—CH₂—, carboxymethyl, sulphonomethyl, where Prot is aprotection group for —OH, —SH and —NH(R^(H)), and Act is an activationgroup for —OH, —SH, and —NH(R^(H)), and R^(H) is hydrogen or C₁₋₆-alkyl.

Examples of protection groups for —OH and —SH groups include substitutedtrityl, such as 4,4′-dimethoxytrityloxy (DMT), 4-monomethoxytrityloxy(MMT); trityloxy, optionally substituted 9-(9-phenyl)xanthenyloxy(pixyl), optionally substituted methoxytetrahydropyranyloxy (mthp);silyloxy, such as trimethylsilyloxy (TMS), triisopropylsilyloxy (TIPS),tert-butyl-dimethylsilyloxy (TBDMS), triethylsilyloxy,phenyldimethylsilyloxy; tert-butylethers; acetals (including two hydroxygroups); acyloxy, such as acetyl or halogen-substituted acetyls, e.g.chloroacetyloxy or fluoroacetyloxy, isobutyryloxy, pivaloyloxy,benzoyloxy and substituted benzoyls, methoxymethyloxy (MOM), benzylethers or substituted benzyl ethers such as 2,6-dichlorobenzyloxy(2,6-Cl₂Bzl). Moreover, when Z or Z* is hydroxyl they may be protectedby attachment to a solid support, optionally through a linker.

Examples of amine protection groups includefluorenylmethoxycarbonylamino (Fmoc), tert-butyloxycarbonylamino (BOC),trifluoroacetylamino, allyloxycarbonylamino (alloc, AOC),Z-benzyloxycarbonylamino (Cbz), substituted benzyloxycarbonylamino, suchas 2-chloro benzyloxycarbonylamino (2-ClZ), monomethoxytritylamino(MMT), dimethoxytritylamino (DMT), phthaloylamino, and9-(9-phenyl)xanthenylamino (pixyl).

The activation group preferably mediates couplings to other residuesand/or nucleotide monomers and after the coupling has been completed theactivation group is typically converted to an internucleoside linkage.Examples of such activation groups include optionally substitutedO-phosphoramidite, optionally substituted O-phosphortriester, optionallysubstituted O-phosphordiester, optionally substituted H-phosphonate, andoptionally substituted O-phosphonate. In the present context, the term“phosphoramidite” means a group of the formula —P(OR^(x))—N(R^(y))₂,wherein R^(x) designates an optionally substituted alkyl group, e.g.methyl, 2-cyanoethyl, or benzyl, and each of R^(y) designates optionallysubstituted alkyl groups, e.g. ethyl or isopropyl, or the group—N(R^(y))₂ forms a morpholino group (—N(CH₂CH₂)₂O). R^(x) preferablydesignates 2-cyanoethyl and the two R^(y) are preferably identical anddesignates isopropyl. Accordingly, a particularly preferredphosphoramidite is N,N-diisopropyl-O-(2-cyanoethyl)phosphoramidite.

The most preferred terminal groups are hydroxy, mercapto and amino, inparticular hydroxy.

Therapy and Pharmaceutical Compositions

As explained initially, the oligonucleotides of the invention willconstitute suitable drugs with improved properties. The design of apotent and safe drug requires the fine-tuning of various parameters suchas affinity/specificity, stability in biological fluids, cellularuptake, mode of action, pharmacokinetic properties and toxicity.

Accordingly, in a further aspect the present invention relates to apharmaceutical composition comprising an oligonucleotide according tothe invention and a pharmaceutically acceptable diluent, carrier oradjuvant. Preferably said carrier is saline of buffered saline.

In a still further aspect the present invention relates to anoligonucleotide according to the present invention for use as amedicament.

As will be understood, dosing is dependent on severity andresponsiveness of the disease state to be treated, and the course oftreatment lasting from several days to several months, or until a cureis effected or a diminution of the disease state is achieved. Optimaldosing schedules can be calculated from measurements of drugaccumulation in the body of the patient. Optimum dosages may varydepending on the relative potency of individual oligonucleotides.Generally it can be estimated based on EC50s found to be effective in invitro and in vivo animal models. In general, dosage is from 0.01 μg to 1g per kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 10 years or by continuousinfusion for hours up to several months. The repetition rates for dosingcan be estimated based on measured residence times and concentrations ofthe drug in bodily fluids or tissues. Following successful treatment, itmay be desirable to have the patient undergo maintenance therapy toprevent the recurrence of the disease state.

Pharmaceutical Compositions

As indicated above, the invention also relates to a pharmaceuticalcomposition, which comprises at least one oligonucleotide of theinvention as an active ingredient. It should be understood that thepharmaceutical composition according to the invention optionallycomprises a pharmaceutical carrier, and that the pharmaceuticalcomposition optionally comprises further compounds, such aschemotherapeutic compounds, anti-inflammatory compounds, antiviralcompounds and/or immuno-modulating compounds.

The oligonucleotides of the invention can be used “as is” or in form ofa variety of pharmaceutically acceptable salts. As used herein, the term“pharmaceutically acceptable salts” refers to salts that retain thedesired biological activity of the herein-identified oligonucleotidesand exhibit minimal undesired toxicological effects. Non-limitingexamples of such salts can be formed with organic amino acid and baseaddition salts formed with metal cations such as zinc, calcium, bismuth,barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium,potassium, and the like, or with a cation formed from ammonia,N,N-dibenzylethylene-diamine, D-glucosamine, tetraethylammonium, orethylenediamine.

In one embodiment of the invention, the oligonucleotide may be in theform of a pro-drug. Oligonucleotides are by virtue negatively chargedions. Due to the lipophilic nature of cell membranes the cellular uptakeof oligonucleotides are reduced compared to neutral or lipophilicequivalents. This polarity “hindrance” can be avoided by using thepro-drug approach (see e.g. Crooke, R. M. (1998) in Crooke, S. T.Antisense research and Application. Springer-Verlag, Berlin, Germany,vol. 131, pp. 103-140).

Pharmaceutically acceptable binding agents and adjuvants may comprisepart of the formulated drug.

Examples of delivery methods for delivery of the therapeutic agentsdescribed herein, as well as details of pharmaceutical formulations,salts, may are well described elsewhere for example in U.S. provisionalapplication 60/838,710 and 60/788,995, which are hereby incorporated byreference, and Danish applications, PA 2006 00615 which is also herebyincorporated by reference.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Delivery ofdrug to tumour tissue may be enhanced by carrier-mediated deliveryincluding, but not limited to, cationic liposomes, cyclodextrins,porphyrin derivatives, branched chain dendrimers, polyethyleniminepolymers, nanoparticles and microspheres (Dass C R. J Pharm Pharmacol2002; 54(1):3-27). The pharmaceutical formulations of the presentinvention, which may conveniently be presented in unit dosage form, maybe prepared according to conventional techniques well known in thepharmaceutical industry. Such techniques include the step of bringinginto association the active ingredients with the pharmaceuticalcarrier(s) or excipient(s). In general the formulations are prepared byuniformly and intimately bringing into association the activeingredients with liquid carriers or finely divided solid carriers orboth, and then, if necessary, shaping the product. The compositions ofthe present invention may be formulated into any of many possible dosageforms such as, but not limited to, tablets, capsules, gel capsules,liquid syrups, soft gels and suppositories. The compositions of thepresent invention may also be formulated as suspensions in aqueous,non-aqueous or mixed media. Aqueous suspensions may further containsubstances which increase the viscosity of the suspension including, forexample, sodium carboxymethylcellulose, sorbitol and/or dextran. Thesuspension may also contain stabilizers. The compounds of the inventionmay also be conjugated to active drug substances, for example, aspirin,ibuprofen, a sulfa drug, an antidiabetic, an antibacterial or anantibiotic.

In another embodiment, compositions of the invention may contain one ormore oligonucleotide compounds, targeted to a first microRNA and one ormore additional oligonucleotide compounds targeted to a second microRNAtarget. Two or more combined compounds may be used together orsequentially.

The compounds disclosed herein are useful for a number of therapeuticapplications as indicated above. In general, therapeutic methods of theinvention include administration of a therapeutically effective amountof an oligonucleotide to a mammal, particularly a human. In a certainembodiment, the present invention provides pharmaceutical compositionscontaining (a) one or more compounds of the invention, and (b) one ormore chemotherapeutic agents. When used with the compounds of theinvention, such chemotherapeutic agents may be used individually,sequentially, or in combination with one or more other suchchemotherapeutic agents or in combination with radiotherapy. Allchemotherapeutic agents known to a person skilled in the art are hereincorporated as combination treatments with compound according to theinvention. Other active agents, such as anti-inflammatory drugs,including but not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, antiviral drugs, and immuno-modulating drugs may alsobe combined in compositions of the invention. Two or more combinedcompounds may be used together or sequentially.

Examples of therapeutic indications which may be treated by thepharmaceutical compositions of the invention:

microRNA Possible medical indications miR-21 Glioblastoma, breast cancermiR-122 hypercholesterolemia, hepatitis C, hemochromatosis miR-19blymphoma and other tumour types miR-155 lymphoma, breast and lung cancermiR-375 diabetes, metabolic disorders miR-181 myoblast differentiation,auto immune disorders

Tumor suppressor gene tropomysin 1 (TPM1) mRNA has been indicated as atarget of miR-21. Myotrophin (mtpn) mRNA has been indicated as a targetof miR 375.

In an even further aspect, the present invention relates to the use ofan oligonucleotide according to the invention for the manufacture of amedicament for the treatment of a disease selected from the groupconsisting of: atherosclerosis, hypercholesterolemia and hyperlipidemia;cancer, glioblastoma, breast cancer, lymphoma, lung cancer; diabetes,metabolic disorders; myoblast differentiation; immune disorders.

The invention further refers to an oligonucleotides according to theinvention for the use in the treatment of from a disease selected fromthe group consisting of: atherosclerosis, hypercholesterolemia andhyperlipidemia; cancer, glioblastoma, breast cancer, lymphoma, lungcancer; diabetes, metabolic disorders; myoblast differentiation; immunedisorders.

The invention provides for a method of treating a subject suffering froma disease or condition selected from from the group consisting of:atherosclerosis, hypercholesterolemia and hyperlipidemia; cancer,glioblastoma, breast cancer, lymphoma, lung cancer; diabetes, metabolicdisorders; myoblast differentiation; immune disorders, the methodcomprising the step of administering an oligonucleotide orpharmaceutical composition of the invention to the subject in needthereof.

Cancer

In an even further aspect, the present invention relates to the use ofan oligonucleotide according to the invention or a conjugate thereof forthe manufacture of a medicament for the treatment of cancer. In anotheraspect, the present invention concerns a method for treatment of, orprophylaxis against, cancer, said method comprising administering anoligonucleotide of the invention or a conjugate thereof, or apharmaceutical composition of the invention to a patient in needthereof.

Such cancers may include lymphoreticular neoplasia, lymphoblasticleukemia, brain tumors, gastric tumors, plasmacytomas, multiple myeloma,leukemia, connective tissue tumors, lymphomas, and solid tumors.

In the use of a compound of the invention or a conjugate thereof for themanufacture of a medicament for the treatment of cancer, said cancer maysuitably be in the form of a solid tumor. Analogously, in the method fortreating cancer disclosed herein said cancer may suitably be in the formof a solid tumor.

Furthermore, said cancer is also suitably a carcinoma. The carcinoma istypically selected from the group consisting of malignant melanoma,basal cell carcinoma, ovarian carcinoma, breast carcinoma, non-smallcell lung cancer, renal cell carcinoma, bladder carcinoma, recurrentsuperficial bladder cancer, stomach carcinoma, prostatic carcinoma,pancreatic carcinoma, lung carcinoma, cervical carcinoma, cervicaldysplasia, laryngeal papillomatosis, colon carcinoma, colorectalcarcinoma and carcinoid tumors. More typically, said carcinoma isselected from the group consisting of malignant melanoma, non-small celllung cancer, breast carcinoma, colon carcinoma and renal cell carcinoma.The malignant melanoma is typically selected from the group consistingof superficial spreading melanoma, nodular melanoma, lentigo malignamelanoma, acral melagnoma, amelanotic melanoma and desmoplasticmelanoma.

Alternatively, the cancer may suitably be a sarcoma. The sarcoma istypically in the form selected from the group consisting ofosteosarcoma, Ewing's sarcoma, chondrosarcoma, malignant fibroushistiocytoma, fibrosarcoma and Kaposi's sarcoma.

Alternatively, the cancer may suitably be a glioma.

A further embodiment is directed to the use of an oligonucleotideaccording to the invention or a conjugate thereof for the manufacture ofa medicament for the treatment of cancer, wherein said medicamentfurther comprises a chemotherapeutic agent selected from the groupconsisting of adrenocorticosteroids, such as prednisone, dexamethasoneor decadron; altretamine (hexalen, hexamethylmelamine (HMM)); amifostine(ethyol); aminoglutethimide (cytadren); amsacrine (M-AMSA); anastrozole(arimidex); androgens, such as testosterone; asparaginase (elspar);bacillus calmette-gurin; bicalutamide (casodex); bleomycin (blenoxane);busulfan (myleran); carboplatin (paraplatin); carmustine (BCNU, BiCNU);chlorambucil (leukeran); chlorodeoxyadenosine (2-CDA, cladribine,leustatin); cisplatin (platinol); cytosine arabinoside (cytarabine);dacarbazine (DTIC); dactinomycin (actinomycin-D, cosmegen); daunorubicin(cerubidine); docetaxel (taxotere); doxorubicin (adriomycin);epirubicin; estramustine (emcyt); estrogens, such as diethylstilbestrol(DES); etopside (VP-16, VePesid, etopophos); fludarabine (fludara);flutamide (eulexin); 5-FUDR (floxuridine); 5-fluorouracil (5-FU);gemcitabine (gemzar); goserelin (zodalex); herceptin (trastuzumab);hydroxyurea (hydrea); idarubicin (idamycin); ifosfamide; IL-2(proleukin, aldesleukin); interferon alpha (intron A, roferon A);irinotecan (camptosar); leuprolide (lupron); levamisole (ergamisole);lomustine (CCNU); mechlorathamine (mustargen, nitrogen mustard);melphalan (alkeran); mercaptopurine (purinethol, 6-MP); methotrexate(mexate); mitomycin-C (mutamucin); mitoxantrone (novantrone); octreotide(sandostatin); pentostatin (2-deoxycoformycin, nipent); plicamycin(mithramycin, mithracin); prorocarbazine (matulane); streptozocin;tamoxifin (nolvadex); taxol (paclitaxel); teniposide (vumon, VM-26);thiotepa; topotecan (hycamtin); tretinoin (vesanoid, all-trans retinoicacid); vinblastine (valban); vincristine (oncovin) and vinorelbine(navelbine). Suitably, the further chemotherapeutic agent is selectedfrom taxanes such as Taxol, Paclitaxel or Docetaxel.

Similarly, the invention is further directed to the use of anoligonucleotide according to the invention or a conjugate thereof forthe manufacture of a medicament for the treatment of cancer, whereinsaid treatment further comprises the administration of a furtherchemotherapeutic agent selected from the group consisting ofadrenocorticosteroids, such as prednisone, dexamethasone or decadron;altretamine (hexalen, hexamethylmelamine (HMM)); amifostine (ethyol);aminoglutethimide (cytadren); amsacrine (M-AMSA); anastrozole(arimidex); androgens, such as testosterone; asparaginase (elspar);bacillus calmette-gurin; bicalutamide (casodex); bleomycin (blenoxane);busulfan (myleran); carboplatin (paraplatin); carmustine (BCNU, BiCNU);chlorambucil (leukeran); chlorodeoxyadenosine (2-CDA, cladribine,leustatin); cisplatin (platinol); cytosine arabinoside (cytarabine);dacarbazine (DTIC); dactinomycin (actinomycin-D, cosmegen); daunorubicin(cerubidine); docetaxel (taxotere); doxorubicin (adriomycin);epirubicin; estramustine (emcyt); estrogens, such as diethylstilbestrol(DES); etopside (VP-16, VePesid, etopophos); fludarabine (fludara);flutamide (eulexin); 5-FUDR (floxuridine); 5-fluorouracil (5-FU);gemcitabine (gemzar); goserelin (zodalex); herceptin (trastuzumab);hydroxyurea (hydrea); idarubicin (idamycin); ifosfamide; IL-2(proleukin, aldesleukin); interferon alpha (intron A, roferon A);irinotecan (camptosar); leuprolide (lupron); levamisole (ergamisole);lomustine (CCNU); mechlorathamine (mustargen, nitrogen mustard);melphalan (alkeran); mercaptopurine (purinethol, 6-MP); methotrexate(mexate); mitomycin-C (mutamucin); mitoxantrone (novantrone); octreotide(sandostatin); pentostatin (2-deoxycoformycin, nipent); plicamycin(mithramycin, mithracin); prorocarbazine (matulane); streptozocin;tamoxifin (nolvadex); taxol (paclitaxel); teniposide (vumon, VM-26);thiotepa; topotecan (hycamtin); tretinoin (vesanoid, all-trans retinoicacid); vinblastine (valban); vincristine (oncovin) and vinorelbine(navelbine). Suitably, said treatment further comprises theadministration of a further chemotherapeutic agent selected fromtaxanes, such as Taxol, Paclitaxel or Docetaxel.

Alternatively stated, the invention is furthermore directed to a methodfor treating cancer, said method comprising administering anoligonucleotide of the invention or a conjugate thereof, or apharmaceutical composition according to the invention to a patient inneed thereof and further comprising the administration of a furtherchemotherapeutic agent. Said further administration may be such that thefurther chemotherapeutic agent is conjugated to the compound of theinvention, is present in the pharmaceutical composition, or isadministered in a separate formulation.

Infectious Diseases

It is contemplated that the compounds of the invention may be broadlyapplicable to a broad range of infectious diseases, such as diphtheria,tetanus, pertussis, polio, hepatitis B, hepatitis C, hemophilusinfluenza, measles, mumps, and rubella.

Hsa-miR122 is indicated in hepatitis C infection and as sucholigonucleotides according to the invention which target miR-122 may beused to treat Hepatitis C infection.

Accordingly, in yet another aspect the present invention relates the useof an oligonucleotide according to the invention or a conjugate thereoffor the manufacture of a medicament for the treatment of an infectiousdisease, as well as to a method for treating an infectious disease, saidmethod comprising administering an oligonucleotide according to theinvention or a conjugate thereof, or a pharmaceutical compositionaccording to the invention to a patient in need thereof.

Inflammatory Diseases

The inflammatory response is an essential mechanism of defense of theorganism against the attack of infectious agents, and it is alsoimplicated in the pathogenesis of many acute and chronic diseases,including autoimmune disorders. In spite of being needed to fightpathogens, the effects of an inflammatory burst can be devastating. Itis therefore often necessary to restrict the symptomatology ofinflammation with the use of anti-inflammatory drugs. Inflammation is acomplex process normally triggered by tissue injury that includesactivation of a large array of enzymes, the increase in vascularpermeability and extravasation of blood fluids, cell migration andrelease of chemical mediators, all aimed to both destroy and repair theinjured tissue.

In yet another aspect, the present invention relates to the use of anoligonucleotide according to the invention or a conjugate thereof forthe manufacture of a medicament for the treatment of an inflammatorydisease, as well as to a method for treating an inflammatory disease,said method comprising administering an oligonucleotide according to theinvention or a conjugate thereof, or a pharmaceutical compositionaccording to the invention to a patient in need thereof.

In one preferred embodiment of the invention, the inflammatory diseaseis a rheumatic disease and/or a connective tissue diseases, such asrheumatoid arthritis, systemic lupus erythematous (SLE) or Lupus,scleroderma, polymyositis, inflammatory bowel disease, dermatomyositis,ulcerative colitis, Crohn's disease, vasculitis, psoriatic arthritis,exfoliative psoriatic dermatitis, pemphigus vulgaris and Sjorgren'ssyndrome, in particular inflammatory bowel disease and Crohn's disease.

Alternatively, the inflammatory disease may be a non-rheumaticinflammation, like bursitis, synovitis, capsulitis, tendinitis and/orother inflammatory lesions of traumatic and/or sportive origin.

Metabolic Diseases

A metabolic disease is a disorder caused by the accumulation ofchemicals produced naturally in the body. These diseases are usuallyserious, some even life threatening. Others may slow physicaldevelopment or cause mental retardation. Most infants with thesedisorders, at first, show no obvious signs of disease. Proper screeningat birth can often discover these problems. With early diagnosis andtreatment, metabolic diseases can often be managed effectively.

In yet another aspect, the present invention relates to the use of anoligonucleotide according to the invention or a conjugate thereof forthe manufacture of a medicament for the treatment of a metabolicdisease, as well as to a method for treating a metabolic disease, saidmethod comprising administering an oligonucleotide according to theinvention or a conjugate thereof, or a pharmaceutical compositionaccording to the invention to a patient in need thereof.

In one preferred embodiment of the invention, the metabolic disease isselected from the group consisting of Amyloidosis, Biotinidase, OMIM(Online Mendelian Inheritance in Man), Crigler Najjar Syndrome,Diabetes, Fabry Support & Information Group, Fatty acid OxidationDisorders, Galactosemia, Glucose-6-Phosphate Dehydrogenase (G6PD)deficiency, Glutaric aciduria, International Organization of GlutaricAcidemia, Glutaric Acidemia Type I, Glutaric Acidemia, Type II, GlutaricAcidemia Type I, Glutaric Acidemia Type-II, F-HYPDRR—FamilialHypophosphatemia, Vitamin D Resistant Rickets, Krabbe Disease, Longchain 3 hydroxyacyl CoA dehydrogenase deficiency (LCHAD), MannosidosisGroup, Maple Syrup Urine Disease, Mitochondrial disorders,Mucopolysaccharidosis Syndromes: Niemann Pick, Organic acidemias, PKU,Pompe disease, Porphyria, Metabolic Syndrome, Hyperlipidemia andinherited lipid disorders, Trimethylaminuria: the fish malodor syndrome,and Urea cycle disorders.

Liver Disorders

In yet another aspect, the present invention relates to the use of anoligonucleotide according to the invention or a conjugate thereof forthe manufacture of a medicament for the treatment of a liver disorder,as well as to a method for treating a liver disorder, said methodcomprising administering an oligonucleotide according to the inventionor a conjugate thereof, or a pharmaceutical composition according to theinvention to a patient in need thereof.

In one preferred embodiment of the invention, the liver disorder isselected from the group consisting of Biliary Atresia, AlegilleSyndrome, Alpha-1 Antitrypsin, Tyrosinemia, Neonatal Hepatitis, andWilson Disease.

Other Uses

The oligonucleotides of the present invention can be utilized for asresearch reagents for diagnostics, therapeutics and prophylaxis. Inresearch, the oligonucleotide may be used to specifically inhibit thesynthesis of target genes in cells and experimental animals therebyfacilitating functional analysis of the target or an appraisal of itsusefulness as a target for therapeutic intervention. In diagnostics theoligonucleotides may be used to detect and quantitate target expressionin cell and tissues by Northern blotting, in-situ hybridisation orsimilar techniques. For therapeutics, an animal or a human, suspected ofhaving a disease or disorder, which can be treated by modulating theexpression of target is treated by administering the oligonucleotidecompounds in accordance with this invention. Further provided aremethods of treating an animal particular mouse and rat and treating ahuman, suspected of having or being prone to a disease or condition,associated with expression of target by administering a therapeuticallyor prophylactically effective amount of one or more of theoligonucleotide compounds or compositions of the invention.

Therapeutic Use of Oligonucleotides Targeting miR-122a

In the examples section, it is demonstrated that a LNA-antimiR™, such asSPC3372, targeting miR-122a reduces plasma cholesterol levels.Therefore, another aspect of the invention is use of the above describedoligonucleotides targeting miR-122a as medicine.

Still another aspect of the invention is use of the above describedoligonucleotides targeting miR-122a for the preparation of a medicamentfor treatment of increased plasma cholesterol levels. The skilled manwill appreciate that increased plasma cholesterol levels is undesireableas it increases the risk of various conditions, e.g. atherosclerosis.

Still another aspect of the invention is use of the above describedoligonucleotides targeting miR-122a for upregulating the mRNA levels ofNrdg3, Aldo A, Bckdk or CD320.

Further Embodiments The Following Embodiments may be Combined with theOther Embodiments of the Invention as Described Herein

1. An oligonucleotide having a length of from 12 to 26 nucleotideshaving a core DNA sequence from positions two to seven or from positionsthree to eight, counting from the 3′ end: acgttt, wherein at least one,such as one, preferably at least two, such as two or three, DNA units insaid sequence have been substituted by their corresponding LNA unit; ora conjugate thereof.

2. An oligonucleotide having a length of from 12 to 26 nucleotideshaving a core DNA sequence from positions two to seven or from positionsthree to eight, counting from the 3′ end: ctcaca, wherein at least one,such as one, preferably at least two, such as two or three, DNA units insaid sequence have been substituted by their corresponding LNA unit; ora conjugate thereof.

3. An oligonucleotide having a length of from 12 to 26 nucleotideshaving a core DNA sequence from positions two to seven or from positionsthree to eight, counting from the 3′ end: ttacga, wherein at least one,such as one, preferably at least two, such as two or three, DNA units insaid sequence have been substituted by their corresponding LNA unit; ora conjugate thereof.

4. An oligonucleotide having a length of from 12 to 26 nucleotideshaving a core DNA sequence from positions two to seven or from positionsthree to eight, counting from the 3′ end: acaagc; wherein at least one,such as one, preferably at least two, such as two or three, DNA units insaid sequence have been substituted by their corresponding LNA unit; ora conjugate thereof.

5. The oligonucleotide according to any one of embodiments 1 to 4 or aconjugate thereof, wherein at least two, such as two or three, DNA unitsfrom positions one to six, two to seven or three to eight, counting fromthe 3′ end, have been substituted by their corresponding LNA unit andwherein the LNA units are separated by at least one DNA unit.

6. The oligonucleotide according to embodiment 5 or a conjugate thereof,wherein the number of consecutive DNA units from positions one to six,two to seven or three to eight, counting from the 3′ end, is at mosttwo.

7. The oligonucleotide according to embodiment 6 or a conjugate thereof,wherein every second nucleotide from positions one to six, two to sevenor three to eight, counting from the 3′ end, is an LNA unit.

8. The oligonucleotide according to embodiment 6 or a conjugate thereof,wherein every third nucleotide from positions one to six, two to sevenor three to eight, counting from the 3′ end, is an LNA unit.

9. The oligonucleotide according to embodiment 6 or a conjugate thereof,wherein the substitution pattern for the nucleotides in positions one tosix, two to seven or three to eight, counting from the 3′ end, isselected from the group consisting of: xxXxxX, xxXxXx, xXxxXx, xXxXxx,XxxXxx, xXxXxX, XxXxXx, XxxXxX, and XxXxxX; wherein “X” denotes an LNAunit and “x” denotes a DNA unit.

10. The oligonucleotide according to embodiment 9 or a conjugatethereof, wherein the substitution pattern for the nucleotides inpositions one to six, two to seven or three to eight, counting from the3′ end, is selected from the group consisting of xxXxxX, xXxxXx, XxxXxx,xXxXxX, and XxXxXx; wherein “X” denotes an LNA unit and “x” denotes aDNA unit.

11. The oligonucleotide according to embodiment 1 or a conjugate thereofhaving a DNA sequence from positions one to seven, two to eight or threeto nine, counting from the 3′ end: acgttta, wherein at least one, suchas one, preferably at least two, such as two, more preferably at leastthree, such as three or four, DNA units in said sequence have beensubstituted by their corresponding LNA unit.

12. The oligonucleotide according to embodiment 2 or a conjugate thereofhaving a DNA sequence from positions one to seven, two to eight or threeto nine, counting from the 3′ end: ctcacac, wherein at least one, suchas one, preferably at least two, such as two, more preferably at leastthree, such as three or four, DNA units in said sequence have beensubstituted by their corresponding LNA unit.

13. The oligonucleotide according to embodiment 3 or a conjugate thereofhaving a DNA sequence from positions one to seven, two to eight or threeto nine, counting from the 3′ end: ttacgat, wherein at least one, suchas one, preferably at least two, such as two, more preferably at leastthree, such as three or four, DNA units in said sequence have beensubstituted by their corresponding LNA unit.

14. The oligonucleotide according to embodiment 4 or a conjugate thereofhaving a DNA sequence from positions one to seven, two to eight or threeto nine, counting from the 3′ end: acaagca, wherein at least one, suchas one, preferably at least two, such as two, more preferably at leastthree, such as three or four, DNA units in said sequence have beensubstituted by their corresponding LNA unit.

15. The oligonucleotide according to any one of embodiments 11 to 14 ora conjugate thereof, wherein at least two, such as two, three or four,DNA units from positions one to seven, two to eight or three to nine,counting from the 3′ end, have been substituted by their correspondingLNA unit and wherein the LNA units are separated by at least one DNAunit.

16. The oligonucleotide according to embodiment 15 or a conjugatethereof, wherein the number of consecutive DNA units from positions oneto seven, two to eight or three to nine, counting from the 3′ end, is atmost two.

17. The oligonucleotide according to embodiment 16 or a conjugatethereof, wherein every second nucleotide from positions one to seven,two to eight or three to nine, counting from the 3′ end, is an LNA unit.

18. The oligonucleotide according to embodiment 16 or a conjugatethereof, wherein every third nucleotide from positions one to seven, twoto eight or three to nine, counting from the 3′ end, is an LNA unit.

19. The oligonucleotide according to embodiment 16 or a conjugatethereof, wherein the substitution pattern for the nucleotides inpositions one to seven, two to eight or three to nine, counting from the3′ end, is selected from the group consisting of xxXxxXx, xxXxXxx,xXxxXxx, xxXxXxX, xXxxXxX, xXxXxxX, xXxXxXx, XxxXxxX, XxxXxXx, XxXxxXx,XxXxXxx, and XxXxXxX;wherein “X” denotes an LNA unit and “x” denotes aDNA unit.

20. The oligonucleotide according to embodiment 19 or a conjugatethereof, wherein the substitution pattern for the nucleotides inpositions one to seven, two to eight or three to nine, counting from the3′ end, is selected from the group consisting of xxXxxXx, xXxxXxx,XxxXxxX, xXxXxXx, XxXxXxX, and XxXxXxx; wherein “X” denotes an LNA unitand “x” denotes a DNA unit.

21. The oligonucleotide according to embodiment 11 or a conjugatethereof having a DNA sequence from positions one to eight, two to nineor three to ten, counting from the 3′ end: acgtttag, wherein at leastone, such as one, preferably at least two, such as two, more preferablyat least three, such as three or four, DNA units in said sequence havebeen substituted by their corresponding LNA unit.

22. The oligonucleotide according to embodiment 12 or a conjugatethereof having a DNA sequence from positions one to eight, two to nineor three to ten, counting from the 3′ end: ctcacact, wherein at leastone, such as one, preferably at least two, such as two, more preferablyat least three, such as three or four, DNA units in said sequence havebeen substituted by their corresponding LNA unit.

23. The oligonucleotide according to embodiment 13 or a conjugatethereof having a DNA sequence from positions one to eight, two to nineor three to ten, counting from the 3′ end: ttacgatt, wherein at leastone, such as one, preferably at least two, such as two, more preferablyat least three, such as three or four, DNA units in said sequence havebeen substituted by their corresponding LNA unit.

24. The oligonucleotide according to embodiment 14 or a conjugatethereof having a DNA sequence from positions one to eight, two to nineor three to ten, counting from the 3′ end: acaagcaa, wherein at leastone, such as one, preferably at least two, such as two, more preferablyat least three, such as three or four, DNA units in said sequence havebeen substituted by their corresponding LNA unit.

25. The oligonucleotide according to any one of embodiments 21 to 24 ora conjugate thereof, wherein at least two, such as two, three or four,DNA units from positions one to eight, two to nine or three to ten,counting from the 3′ end, have been substituted by their correspondingLNA unit and wherein the LNA units are separated by at least one DNAunit.

26. The oligonucleotide according to embodiment 25 or a conjugatethereof, wherein the number of consecutive DNA units from positions oneto eight, two to nine or three to ten, counting from the 3′ end, is atmost two.

27. The oligonucleotide according to embodiment 26 or a conjugatethereof, wherein every second nucleotide from positions one to eight,two to nine or three to ten, counting from the 3′ end, is an LNA unit.

28. The oligonucleotide according to embodiment 26 or a conjugatethereof, wherein every third nucleotide from positions one to eight, twoto nine or three to ten, counting from the 3′ end, is an LNA unit.

29. The oligonucleotide according to embodiment 26 or a conjugatethereof, wherein the substitution pattern for the nucleotides inpositions one to eight, two to nine or three to ten, counting from the3′ end, is selected from the group consisting of xxXxxXxx, xxXxxXxX,xxXxXxxX, xxXxXxXx, xXxxXxxX, xXxxXxXx, xXxXxxXx, xXxXxXxx, XxxXxxXx,XxxXxXxx, XxXxxXxx, xXxXxXxX, XxXxXxxX, XxXxxXxX, XxxXxXxX, andXxXxXxXx; wherein “X” denotes an LNA unit and “x” denotes a DNA unit.

30. The oligonucleotide according to embodiment 29 or a conjugatethereof, wherein the substitution pattern for the nucleotides inpositions one to eight, two to nine or three to ten, counting from the3′ end, is selected from the group consisting of xxXxxXxx, xXxxXxxX,XxxXxxXx, xXxXxXxX, XxXxXxXx, and XxXxXxxX; wherein “X” denotes an LNAunit and “x” denotes a DNA unit.

31. The oligonucleotide according to embodiment 21 or a conjugatethereof having a DNA sequence from positions one to nine, two to ten orthree to eleven, counting from the 3′ end: acgtttagg, wherein at leastone, such as one, preferably at least two, such as two, more preferablyat least three, such as three, even more preferably at least four, suchas four or five, DNA units in said sequence have been substituted bytheir corresponding LNA unit.

32. The oligonucleotide according to embodiment 22 or a conjugatethereof having a DNA sequence from positions one to nine, two to ten orthree to eleven, counting from the 3′ end: ctcacactg, wherein at leastone, such as one, preferably at least two, such as two, more preferablyat least three, such as three, even more preferably at least four, suchas four or five, DNA units in said sequence have been substituted bytheir corresponding LNA unit.

33. The oligonucleotide according to embodiment 23 or a conjugatethereof having a DNA sequence from positions one to nine, two to ten orthree to eleven, counting from the 3′ end: ttacgatta, wherein at leastone, such as one, preferably at least two, such as two, more preferablyat least three, such as three, even more preferably at least four, suchas four or five, DNA units in said sequence have been substituted bytheir corresponding LNA unit.

34. The oligonucleotide according to embodiment 24 or a conjugatethereof having a DNA sequence from positions one to nine, two to ten orthree to eleven, counting from the 3′ end: acaagcaag, wherein at leastone, such as one, preferably at least two, such as two, more preferablyat least three, such as three, even more preferably at least four, suchas four or five, DNA units in said sequence have been substituted bytheir corresponding LNA unit.

35. The oligonucleotide according to any one of embodiments 21 to 24 ora conjugate thereof, wherein at least two, such as two, three, four orfive, DNA units from positions one to nine, two to ten or three toeleven, counting from the 3′ end, have been substituted by theircorresponding LNA unit and wherein the LNA units are separated by atleast one DNA unit.

36. The oligonucleotide according to embodiment 35 or a conjugatethereof, wherein the number of consecutive DNA units from positions oneto nine, two to ten or three to eleven, counting from the 3′ end, is atmost two.

37. The oligonucleotide according to embodiment 36 or a conjugatethereof, wherein every second nucleotide from positions one to nine, twoto ten or three to eleven, counting from the 3′ end, is an LNA unit.

38. The oligonucleotide according to embodiment 36 or a conjugatethereof, wherein every third nucleotide from positions one to nine, twoto ten or three to eleven, counting from the 3′ end, is an LNA unit.

39. The oligonucleotide according to embodiment 36 or a conjugatethereof, wherein the substitution pattern for the nucleotides inpositions one to nine, two to ten or three to eleven, counting from the3′ end, is selected from the group consisting of xxXxxXxxX, xxXxxXxXx,xxXxXxxXx, xxXxXxXxx, xXxxXxxXx, xXxxXxXxx, xXxXxxXxx, XxxXxxXxx,xxXxXxXxX, xXxxXxXxX, xXxXxxXxX, xXxXxXxxX, XxxXxxXxX, XxxXxXxxX,XxXxxXxxX, XxxXxXxXx, XxXxxXxXx, XxXxXxxXx, XxXxXxXxx, and XxXxXxXxX;wherein “X” denotes an LNA unit and “x” denotes a DNA unit.

40. The oligonucleotide according to any of the preceding embodiments ora conjugate thereof, wherein said nucleotide has a length of from 12 to24 nucleotides, such as a length of from 12 to 22 nucleotides,preferably a length of from 12 to 20 nucleotides, such as a length offrom 12 to 19 nucleotides, more preferably a length of from 12 to 18nucleotides, such as a length of from 12 to 17 nucleotides, even morepreferably a length of from 12 to 16 nucleotides.

41. The oligonucleotide according to embodiment 1 having a sequenceselected from the group consisting of tg^(Me)CatGgaTttGca^(Me)Ca,tg^(Me)CatGgaTttGca ^(Me)C, ^(Me)CatGgaTttGca^(Me)C, tGcAtGgAtTtGcAc,cAtGgAtTtGcAc, ^(Me)CatGGatTtGcA^(Me)C, Tg^(Me)CatGGatTtGcA^(Me)C, andTg^(Me)CaTgGaTTtGcACa; wherein a lowercase letter identifies thenitrogenous base of a DNA unit and an uppercase letter identifies thenitrogenous base of an LNA unit; or a conjugate thereof. (SEQ IDs NO82-89)

42. The oligonucleotide according to embodiment 2 having a sequenceselected from the group consisting of c^(Me)CatTgtCacAct^(Me)Cca,c^(Me)CatTgtAacTct^(Me)Cca, ccAttGtcAca^(Me)Ctc^(Me)Ca,c^(Me)CatTgt^(Me)CacAct^(Me)Cc, atTgt^(Me)CacAct^(Me)Cc,ccAttGtcAca^(Me)Ctc^(Me)C, AttGtcAca^(Me)Ctc^(Me)C,aTtGt^(Me)CaCa^(Me)Ct^(Me)Cc, AttGTca^(Me)Ca^(Me)Ct^(Me)C^(Me)C,^(Me)CcAttGTca^(Me)Ca^(Me)Ct^(Me)C^(Me)C, ^(Me)CcaTtgTcacActc^(Me)Ca,and ^(Me)C^(Me)CAttgtcacacT^(Me)C^(Me)Ca; wherein a lowercase letteridentifies the nitrogenous base of a DNA unit and an uppercase letteridentifies the nitrogenous base of an LNA unit; or a conjugate thereof.(SEQ IDs NO 90-101)

43. The oligonucleotide according to embodiment 3 having a sequenceselected from the group consisting of t^(Me)CacGatTag^(Me)CatTaa,aTca^(Me)CgaTtaGcaTta, TcAcGaTtAg^(Me)CaTtAa, AtcAcGaTtAg^(Me)CaTta;wherein a lowercase letter identifies the nitrogenous base of a DNA unitand an uppercase letter identifies the nitrogenous base of an LNA unit;or a conjugate thereof. (SEQ IDs NO 102-105).

44. The oligonucleotide according to embodiment 4 having a sequenceselected from the group consisting of gAgc^(Me)CgaAcgAacAa,gc^(Me)CgaAcgAacAa, GaGc^(Me)CgAa^(Me)CgAa^(Me)CaA, andGc^(M)eCgAa^(Me)CgAa^(Me)CaA; wherein a lowercase letter identifies thenitrogenous base of a DNA unit and an uppercase letter identifies thenitrogenous base of an LNA unit; or a conjugate thereof. (SEQ IDs NO106-109).

45. The oligonucleotide according to any of the preceding embodiments ora conjugate thereof, wherein the oligonucleotide comprises at least oneinternucleoside linkage group which differs from phosphodiester.

46. The oligonucleotide according to embodiment 45 or a conjugatethereof, wherein said internucleoside linkage group, which differs fromphosphodiester, is phosphorothioate.

47. The oligonucleotide according to embodiment 46 or a conjugatethereof, wherein all internucleoside linkage groups arephosphorothioate.

48. The oligonucleotide according to any of the preceding embodiments ora conjugate thereof, wherein said LNA units are independently selectedfrom the group consisting of thio-LNA units, amino-LNA units and oxy-LNAunits.

49. The oligonucleotide according to embodiment 48 or a conjugatethereof, wherein said LNA units are in the beta-D-form.

50. The oligonucleotide according to embodiment 48 or a conjugatethereof, wherein said LNA units are oxy-LNA units in the beta-D-form.

51. The oligonucleotide according to any of the preceding embodiments ora conjugate thereof for use as a medicament.

52. A pharmaceutical composition comprising an oligonucleotide accordingto any of embodiments 1-50 or a conjugate thereof and a pharmaceuticallyacceptable carrier.

53. The composition according to embodiment 52, wherein said carrier issaline or buffered saline.

54. Use of an oligonucleotide according to any of embodiments 1-50 or aconjugate thereof, or a composition according to embodiment 52 for themanufacture of a medicament for the treatment of cancer.

55. A method for the treatment of cancer, comprising the step ofadministering an oligonucleotide according to any of embodiment 1-50 ora conjugate thereof, or a composition according to embodiment 52.

56. Use of an oligonucleotide according to any of embodiments 1-50 or aconjugate thereof, or a composition according to embodiment 52 for thepreparation of a medicament for treatment of increased plasmacholesterol levels.

57. Use of an oligonucleotide according to any of embodiments 1-50 or aconjugate thereof, or a composition according to embodiment 52 forupregulating the mRNA levels of Nrdg3, Aldo A, Bckdk or CD320.

Experimental

Example 1 Monomer Synthesis

The LNA monomer building blocks and derivatives thereof were preparedfollowing published procedures and references cited therein, see, e.g.WO 03/095467 A1 and D. S. Pedersen, C. Rosenbohm, T. Koch (2002)Preparation of LNA Phosphoramidites, Synthesis 6, 802-808.

Example 2 Oligonucleotide Synthesis

Oligonucleotides were synthesized using the phosphoramidite approach onan Expedite 8900/MOSS synthesizer (Multiple Oligonucleotide SynthesisSystem) at 1 μmol or 15 μmol scale. For larger scale synthesis an ÄktaOligo Pilot (GE Healthcare) was used. At the end of the synthesis(DMT-on), the oligonucleotides were cleaved from the solid support usingaqueous ammonia for 1-2 hours at room temperature, and furtherdeprotected for 4 hours at 65° C. The oligonucleotides were purified byreverse phase HPLC (RP-HPLC). After the removal of the DMT-group, theoligonucleotides were characterized by AE-HPLC, RP-HPLC, and CGE and themolecular mass was further confirmed by ESI-MS. See below for moredetails.

Preparation of the LNA-Solid Support:

Preparation of the LNA Succinyl Hemiester

5′-O-Dmt-3′-hydroxy-LNA monomer (500 mg), succinic anhydride (1.2 eq.)and DMAP (1.2 eq.) were dissolved in DCM (35 mL). The reaction wasstirred at room temperature overnight. After extractions with NaH₂PO₄0.1 M pH 5.5 (2×) and brine (1×), the organic layer was further driedwith anhydrous Na₂SO₄ filtered and evaporated. The hemiester derivativewas obtained in 95% yield and was used without any further purification.

Preparation of the LNA-Support

The above prepared hemiester derivative (90 μmol) was dissolved in aminimum amount of DMF, DIEA and pyBOP (90 μmol) were added and mixedtogether for 1 min. This pre-activated mixture was combined withLCAA-CPG (500 Å, 80-120 mesh size, 300 mg) in a manual synthesizer andstirred. After 1.5 hours at room temperature, the support was filteredoff and washed with DMF, DCM and MeOH. After drying, the loading wasdetermined to be 57 μmol/g (see Tom Brown, Dorcas J. S. Brown. Modernmachine-aided methods of oligodeoxyribonucleotide synthesis. In: F.Eckstein, editor. Oligonucleotides and Analogues A Practical Approach.Oxford: IRL Press, 1991: 13-14).

Elongation of the Oligonucleotide

The coupling of phosphoramidites (A(bz), G(ibu), 5-methyl-C(bz)) orT-13-cyanoethyl-phosphoramidite) is performed by using a solution of 0.1M of the 5′-O-DMT-protected amidite in acetonitrile and DCI(4,5-dicyanoimidazole) in acetonitrile (0.25 M) as activator. Thethiolation is carried out by using xanthane chloride (0.01 M inacetonitrile:pyridine 10%). The rest of the reagents are the onestypically used for oligonucleotide synthesis.

Purification by RP-HPLC:

Column: Xterra RP₁₈

Flow rate: 3 mL/min

Buffers: 0.1 M ammonium acetate pH 8 and acetonitrile

Abbreviations:

DMT: Dimethoxytrityl

DCI: 4,5-Dicyanoimidazole

DMAP: 4-Dimethylaminopyridine

DCM: Dichloromethane

DMF: Dimethylformamide

THF: Tetrahydrofurane

DIEA: N,N-diisopropylethylamine

PyBOP: Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphoniumhexafluorophosphate

Bz: Benzoyl

Ibu: Isobutyryl

Example 3 Design of the LNA Anti-miR Oligonucleotides and MeltingTemperatures

Target microRNA:

SEQ ID NO: 1 miR-122a: 5′-uggagugugacaaugguguuugu-3′ (SEQ ID NO: 1reverse orientation) miR-122a 3′ to 5′: 3′-uguuugugguaacagugugaggu-5′

TABLE 1 LNA anti-miR oligonucleotide sequences and T_(m): SEQ ID Tm NO:Oligo ID SED ID Sequence: (° C.) 2 SPC3370 XxxX SEQ ID 565′-cCatTgtCacActCca- PS 75 design 3′ backbone 3 SPC3372 XxxX SEQ ID 575′-ccAttGtcAcaCtcCa- PS 69 design 3′ backbone 4 SPC3375 Gapmer SEQ ID 585′- PS 69 CCAttgtcacacTCCa-3′ backbone 5 SPC3549 15-mer SEQ ID 595′-CcAttGTcaCaCtCC- PS 78 3′ backbone 6 SPC3550 mismatch SEQ ID 605′-CcAttCTgaCcCtAC- PS 32 control 3′ backbone 7 SPC3373 mismatch SEQ ID61 5′-ccAttGtcTcaAtcCa- PS 46 control 3′ backbone 8 SPC3548 13-mer SEQID 62 5′-AttGTcaCaCtCC-3′ PS backbone

lower case: DNA, uppercase: LNA (all LNA C were methylated), underlined:mismatch

The melting temperatures were assessed towards the mature miR-122asequence, using a synthetic miR-122a RNA oligonucleotide withphosphorothioate linkaged.

The LNA anti-miR/miR-122a oligo duplex was diluted to 3 μM in 500 μlRNase free H₂0, which was then mixed with 500 μl 2× dimerization buffer(final oligo/duplex conc. 1.5 μM, 2× Tm buffer: 200 mM NaCl, 0.2 mMEDTA, 20 mM NaP, pH 7.0, DEPC treated to remove RNases). The mix wasfirst heated to 95 degrees for 3 minutes, then allowed to cool at roomtemperature (RT) for 30 minutes.

Following RT incubation T_(m) was measured on Lambda 40 UV/VISSpectrophotometer with peltier temperature progammer PTP6 using PETemplab software (Perkin Elmer). The temperature was ramped up from 20°C. to 95° C. and then down again to 20° C., continuously recordingabsorption at 260 nm. First derivative and local maximums of both themelting and annealing was used to assess melting/annealing point(T_(m)), both should give similar/same T_(m) values. For the firstderivative 91 points was used to calculate the slope.

By substituting the antimir oligonucleotide and the complementary RNAmolecule, the above assay can be used to determine the T_(m) of otheroligonucleotides such as the oligonucleotides according to theinvention.

However, in one embodiment the T_(m) may be made with a complementaryDNA (phosphorothioate linkages) molecule. Typically the T_(m) measuredagainst a DNA complementary molecule is about 10° C. lower than theT_(m) with an equivalent RNA complement. The T_(m) measured using theDNA complement may therefore be used in cases where the duplex has avery high T_(m).

Melting temperature (T_(m)) measurements:

T_(m) oligo to miR-122 RNA complement SPC3372 + miR-122a, RNA 69° C.SPC3648 + miR-122a, RNA 74° C. SPC3649 + miR-122a, RNA 79° C. oligo toDNA complement SPC3372 + 122R, DNA 57° C. SPC3649 + 122R, DNA 66° C.

It is recognised that for oligonucleotides with very high T_(m), theabove T_(m) assays may be insufficient to determine the T_(m). In suchan instance the use of a phosphorothioated DNA complementary moleculemay further lower the T_(m).

The use of formamide is routine in the analysis of oligonucleotidehybridisation (see Hutton 1977, NAR 4 (10) 3537-3555). In the aboveassay the inclusion of 15% formamide typically lowers the T_(m) by about9° C., and the inclusion of 50% formamide typically lowers the T_(m) byabout 30° C. Using these ratios, it is therefore possible to determinethe comparative T_(m) of an oligonucleotide against its complementaryRNA (phosphodiester) molecule, even when the T_(m) of the duplex is, forexample higher than 95° C. (in the absence of formamide).

For oligonucleotides with a very high T_(m), an alternative method ofdetermining the T_(m), is to make titrations and run it out on a gel tosee single strand versus duplex and by those concentrations and ratiosdetermine Kd (the dissociation constant) which is related to deltaG andalso T_(m).

Example 4 Stability of LNA Oligonucleotides in Human or Rat Plasma

LNA oligonucleotide stability was tested in plasma from human or rats(it could also be mouse, monkey or dog plasma). In 45 μl plasma, 5 μlLNA oligonucleotide is added (at a final concentration of 20 μM). TheLNA oligonucleotides are incubated in plasma for times ranging from 0 to96 hours at 37° C. (the plasma is tested for nuclease activity up to 96hours and shows no difference in nuclease cleavage-pattern).

At the indicated time the sample were snap frozen in liquid nitrogen. 2μL (equals 40 pmol) LNA oligonucleotide in plasma was diluted by adding15 μL of water and 3 μL 6× loading dye (Invitrogen). As marker a 10 byladder (Invitrogen, USA 10821-015) is used. To 1 μl ladder, 1 μl 6×loading and 4 μl water is added. The samples are mixed, heated to 65° C.for 10 min and loaded to a pre-run gel (16% acrylamide, 7 M UREA, 1×TBE, pre-run at 50 Watt for 1 h) and run at 50-60 Watt for 2½ hours.Subsequently, the gel is stained with 1× SyBR gold (molecular probes) in1× TBE for 15 min. The bands were visualised using a phosphoimager fromBioRad.

Example 5 In vitro Model: Cell Culture

The effect of LNA oligonucleotides on target nucleic acid expression(amount) can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. Target can beexpressed endogenously or by transient or stable transfection of anucleic acid encoding said nucleic acid.

The expression level of target nucleic acid can be routinely determinedusing, for example, Northern blot analysis (including microRNAnorthern), Quantitative PCR (including microRNA qPCR), Ribonucleaseprotection assays. The following cell types are provided forillustrative purposes, but other cell types can be routinely used,provided that the target is expressed in the cell type chosen.

Cells were cultured in the appropriate medium as described below andmaintained at 37° C. at 95-98% humidity and 5% CO₂. Cells were routinelypassaged 2-3 times weekly.

15PC3: The human prostate cancer cell line 15PC3 was kindly donated byDr. F. Baas, Neurozintuigen Laboratory, AMC, The Netherlands and wascultured in DMEM (Sigma)+10% fetal bovine serum (FBS)+GlutamaxI+gentamicin.

PC3: The human prostate cancer cell line PC3 was purchased from ATCC andwas cultured in F12 Coon's with glutamine (Gibco)+10% FBS+gentamicin.

518A2: The human melanoma cancer cell line 518A2 was kindly donated byDr. B. Jansen, Section of experimental Oncology, Molecular Pharmacology,Department of Clinical Pharmacology, University of Vienna and wascultured in DMEM (Sigma)+10% fetal bovine serum (FBS)+GlutamaxI+gentamicin.

HeLa: The cervical carcinoma cell line HeLa was cultured in MEM (Sigma)containing 10% fetal bovine serum gentamicin at 37° C., 95% humidity and5% CO₂.

MPC-11: The murine multiple myeloma cell line MPC-11 was purchased fromATCC and maintained in DMEM with 4 mM Glutamax+10% Horse Serum.

DU-145: The human prostate cancer cell line DU-145 was purchased fromATCC and maintained in RPMI with Glutamax+10% FBS.

RCC-4+/− VHL: The human renal cancer cell line RCC4 stably transfectedwith plasmid expressing VHL or empty plasmid was purchased from ECACCand maintained according to manufacturers instructions.

786-0: The human renal cell carcinoma cell line 786-0 was purchased fromATCC and maintained according to manufacturers instructions

HUVEC: The human umbilical vein endothelial cell line HUVEC waspurchased from Camcrex and maintained in EGM-2 medium.

K562: The human chronic myelogenous leukaemia cell line K562 waspurchased from ECACC and maintained in RPMI with Glutamax+10% FBS.U87MG: The human glioblastoma cell line U87MG was purchased from ATCCand maintained according to the manufacturers instructions.

B16: The murine melanoma cell line B16 was purchased from ATCC andmaintained according to the manufacturers instructions.

LNCap: The human prostate cancer cell line LNCap was purchased from ATCCand maintained in RPMI with Glutamax+10% FBS

Huh-7: Human liver, epithelial like cultivated in Eagles MEM with 10%FBS, 2 mM Glutamax I, 1× non-essential amino acids, Gentamicin 25 μg/ml

L428: (Deutsche Sammlung fur Mikroorganismen (DSM, Braunschwieg,Germany)): Human B cell lymphoma maintained in RPMI 1640 supplementedwith 10% FCS, L-glutamine and antibiotics.

L1236: (Deutsche Sammlung für Mikroorganismen (DSM, Braunschwieg,Germany)): Human B cell lymphoma maintained in RPMI 1640 supplementedwith 10% FCS, L-glutamine and antibiotics.

Example 6 In vitro Model: Treatment with LNA Anti-miR AntisenseOligonucleotide

The miR-122a expressing cell line Huh-7 was transfected with LNAanti-miRs at 1 and 100 nM concentrations according to optimizedlipofectamine 2000 (LF2000, Invitrogen) protocol (as follows).

Huh-7 cells were cultivated in Eagles MEM with 10% FBS, 2 mM Glutamax I,1× non-essential amino acids, Gentamicin 25 μg/ml. The cells were seededin 6-well plates (300000 cells per well), in a total vol. of 2.5 ml theday before transfection. At the day of transfection a solutioncontaining LF2000 diluted in Optimem (Invitrogen) was prepared (1.2 mloptimem+3.75 μl LF2000 per well, final 2.5 μg LF2000/ml, final tot vol1.5 ml).

LNA Oligonucleotides (LNA anti-miRs) were also diluted in optimem. 285μl optimem+15 μl LNA oligonucleotide (10 μM oligonucleotide stock forfinal concentration 100 nM and 0.1 μM for final concentration 1 nM)Cells were washed once in optimem then the 1.2 ml optimem/LF2000 mixwere added to each well. Cells were incubated 7 min at room temperaturein the LF2000 mix where after the 300 μl oligonucleotide optimemsolution was added.

Cell were further incubated for four hours with oligonucleotide andlipofectamine2000 (in regular cell incubator at 37° C., 5% CO2). Afterthese four hours the medium/mix was removed and regular complete mediumwas added. Cells were allowed to grow for another 20 hours. Cells wereharvested in Trizol (Invitrogen) 24 hours after transfection. RNA wasextracted according to a standard Trizol protocol according to themanufacturer's instructions (Invitrogen), especially to retain themicroRNA in the total RNA extraction.

Example 7 In vitro and in vivo Model: Analysis of OligonucleotideInhibition of miR Expression by MicroRNA Specific Quantitative PCR

miR-122a levels in the RNA samples were assessed on an ABI 7500 Fastreal-time PCR instrument (Applied Biosystems, USA) using a miR-122aspecific qRT-PCR kit, mirVana (Ambion, USA) and miR-122a primers(Ambion, USA). The procedure was conducted according to themanufacturers protocol.

Results:

The miR-122a-specific new LNA anti-miR oligonucleotide design (ieSPC3349 (also referred to as SPC3549)), was more efficient in inhibitingmiR-122a at 1 nM compared to previous design models, including“every-third” and “gap-mer” (SPC3370, SPC3372, SPC3375) motifs were at100 nM. The mismatch control was not found to inhibit miR-122a(SPC3350). Results are shown in FIG. 1.

Example 8 Assessment of LNA Antago-mir Knock-Down Specificity UsingmiRNA Microarray Expression Profiling

A) RNA Labeling for miRNA Microarray Profiling

Total RNA was extracted using Trizol reagent (Invitrogen) and 3′ endlabeled using T4 RNA ligase and Cy3- or Cy5-labeled RNA linker(5′-PO4-rUrUrU-Cy3/dT-3′ or 5′-PO4-rUrUrU-Cy5/dT-3′). The labelingreactions contained 2-5 μg total RNA, 15 μM RNA linker, 50 mM Tris-HCl(pH 7.8), 10 mM MgCl2, 10 mM DTT, 1 mM ATP, 16% polyethylene glycol and5 unit T4 RNA ligase (Ambion, USA) and were incubated at 30° C. for 2hours followed by heat inactivation of the T4 RNA ligase at 80° C. for 5minutes.

B) Microarray Hybridization and Post-Hybridization Washes

LNA-modified oligonucleotide capture probes comprising probes for allannotated miRNAs annotated from mouse (Mus musculus) and human (Homosapiens) in the miRBase MicroRNA database Release 7.1 including a set ofpositive and negative control probes were purchased from Exiqon (Exiqon,Denmark) and used to print the microarrays for miRNA profiling. Thecapture probes contain a 5′-terminal C6-amino modified linker and weredesigned to have a Tm of 72° C. against complementary target miRNAs byadjustment of the LNA content and length of the capture probes. Thecapture probes were diluted to a final concentration of 10 μM in 150 mMsodium phosphate buffer (pH 8.5) and spotted in quadruplicate ontoCodelink slides (Amersham Biosciences) using the MicroGrid II arrayerfrom BioRobotics at 45% humidity and at room temperature. Spotted slideswere post-processed as recommended by the manufacturer.

Labeled RNA was hybridized to the LNA microarrays overnight at 65° C. ina hybridization mixture containing 4×SSC, 0.1% SDS, 1 μg/μl HerringSperm DNA and 38% formamide. The hybridized slides were washed threetimes in 2×SSC, 0.025% SDS at 65° C., followed by three times in0.08×SSC and finally three times in 0.4×SSC at room temperature.

C) Array Scanning, Image Analysis and Data Processing

The microarrays were scanned using the ArrayWorx scanner (AppliedPrecision, USA) according to the manufacturer's recommendations. Thescanned images were imported into TIGR Spotfinder version 3.1 (Saeed etal., 2003) for the extraction of mean spot intensities and median localbackground intensities, excluding spots with intensities below medianlocal background+4× standard deviations. Background-correlatedintensities were normalized using variance stabilizing normalizationpackage version 1.8.0 (Huber et al., 2002) for R (www.r-project.org).Intensities of replicate spots were averaged using Microsoft Excel.Probes displaying a coefficient of variance>100% were excluded fromfurther data analysis.

Example 9 Detection of MicroRNAs by in Situ Hybridization Detection ofMicroRNAs in Formalin-Fixed Paraffin-Embedded Tissue Sections by in SituHybridization

A) Preparation of the Formalin-Fixed, Paraffin-Embedded Sections for inSitu Hybridization

Archival paraffin-embedded samples are retrieved and sectioned at 5 to10 mm sections and mounted in positively-charged slides using floatationtechnique. Slides are stored at 4° C. until the in situ experiments areconducted.

B) In Situ Hybridization

Sections on slides are deparaffinized in xylene and then rehydratedthrough an ethanol dilution series (from 100% to 25%). Slides aresubmerged in DEPC-treated water and subject to HCl and 0.2% Glycinetreatment, re-fixed in 4% paraformaldehyde and treated with aceticanhydride/triethanolamine; slides are rinsed in several washes of 1× PBSin-between treatments. Slides are pre-hybridized in hyb solution (50%formamide, 5×SSC, 500 mg/mL yeast tRNA, 1× Denhardt) at 50° C. for 30min. Then, 3 pmol of a FITC-labeled LNA probe (Exiqon, Denmark)complementary to each selected miRNA is added to the hyb. solution andhybridized for one hour at a temperature 20-25° C. below the predictedTm of the probe (typically between 45-55° C. depending on the miRNAsequence). After washes in 0.1× and 0.5× SCC at 65° C., a tyramidesignal amplification reaction was carried out using the GenpointFluorescein (FITC) kit (DakoCytomation, Denmark) following the vendor'srecommendations. Finally, slides are mounted with Prolong Gold solution.Fluorescence reaction is allowed to develop for 16-24 hr beforedocumenting expression of the selected miRNA using an epifluorescencemicroscope.

Detection of microRNAs by Whole-Mount in Situ Hybridization ofZebrafish, Xenopus and Mouse Embryos.

All washing and incubation steps are performed in 2 ml eppendorf tubes.Embryos are fixed overnight at 4 oC. in 4% paraformaldehyde in PBS andsubsequently transferred through a graded series (25% MeOH in PBST (PBScontaining 0.1% Tween-20), 50% MeOH in PBST, 75% MeOH in PBST) to 100%methanol and stored at −20 oC up to several months. At the first day ofthe in situ hybridization embryos are rehydrated by successiveincubations for 5 min in 75% MeOH in PBST, 50% MeOH in PBST, 25% MeOH inPBST and 100% PBST (4×5 min).

Fish, mouse and Xenopus embryos are treated with proteinaseK (10 μg/mlin PBST) for 45 min at 37 oC, refixed for 20 min in 4% paraformaldehydein PBS and washed 3×5 min with PBST. After a short wash in water,endogenous alkaline phosphatase activity is blocked by incubation of theembryos in 0.1 M tri-ethanolamine and 2.5% acetic anhydride for 10 min,followed by a short wash in water and 5×5 min washing in PBST. Theembryos are then transferred to hybridization buffer (50% Formamide,5×SSC, 0.1% Tween, 9.2 mM citric acid, 50 ug/ml heparin, 500 ug/ml yeastRNA) for 2-3 hour at the hybridization temperature. Hybridization isperformed in fresh pre-heated hybridization buffer containing 10 nM of3′ DIG-labeled LNA probe (Roche Diagnostics) complementary to eachselected miRNA. Post-hybridization washes are done at the hybridizationtemperature by successive incubations for 15 min in HM- (hybridizationbuffer without heparin and yeast RNA), 75% HM-/25% 2×SSCT (SSCcontaining 0.1% Tween-20), 50% HM-/50% 2× SSCT, 25% HM-/75% 2× SSCT,100% 2×SSCT and 2×30 min in 0.2×SSCT.

Subsequently, embryos are transferred to PBST through successiveincubations for 10 min in 75% 0.2×SSCT/25% PBST, 50% 0.2×SSCT/50% PBST,25% 0.2×SSCT/75% PBST and 100% PBST. After blocking for 1 hour inblocking buffer (2% sheep serum/2 mg:ml BSA in PBST), the embryos areincubated overnight at 4° C. in blocking buffer containing anti-DIG-APFAB fragments (Roche, 1/2000). The next day, zebrafish embryos arewashed 6×15 min in PBST, mouse and X. tropicalis embryos are washed 6×1hour in TBST containing 2 mM levamisole and then for 2 days at 4 oC withregular refreshment of the wash buffer.

After the post-antibody washes, the embryos are washed 3×5 min instaining buffer (100 mM tris HCl pH9.5, 50 mM MgCl2, 100 mM NaCl, 0.1%tween 20). Staining was done in buffer supplied with 4.5 μl/ml NBT(Roche, 50 mg/ml stock) and 3.5 μl/ml BCIP (Roche, 50 mg/ml stock). Thereaction is stopped with 1 mM EDTA in PBST and the embryos are stored at4° C. The embryos are mounted in Murray's solution (2:1benzylbenzoate:benzylalcohol) via an increasing methanol series (25%MeOH in PBST, 50% MeOH in PBST, 75% MeOH in PBST, 100% MeOH) prior toimaging.

Example 10 In vitro Model: Isolation and Analysis of mRNA Expression(Total RNA Isolation and cDNA Synthesis for mRNA Analysis)

Total RNA was isolated either using RNeasy mini kit (Qiagen) or usingthe Trizol reagent (Invitrogen). For total RNA isolation using RNeasymini kit (Qiagen), cells were washed with PBS, and Cell Lysis Buffer(RTL, Qiagen) supplemented with 1% mercaptoethanol was added directly tothe wells. After a few minutes, the samples were processed according tomanufacturer's instructions.

For in vivo analysis of mRNA expression tissue samples were firsthomogenised using a Retsch 300MM homogeniser and total RNA was isolatedusing the Trizol reagent or the RNeasy mini kit as described by themanufacturer.

First strand synthesis (cDNA from mRNA) was performed using eitherOmniScript Reverse Transcriptase kit or M-MLV Reverse transcriptase(essentially described by manufacturer (Ambion)) according to themanufacturer's instructions (Qiagen). When using OmniScript ReverseTranscriptase 0.5 μg total RNA each sample, was adjusted to 12 μl andmixed with 0.2 μl poly (dT)₁₂₋₁₈ (0.5 μg/μl) (Life Technologies), 2 μldNTP mix (5 mM each), 2 μl 10× RT buffer, 0.5 μl RNAguard™ RNaseInhibitor (33 units/ml, Amersham) and 1 μl OmniScript ReverseTranscriptase followed by incubation at 37° C. for 60 min. and heatinactivation at 93° C. for 5 min.

When first strand synthesis was performed using random decamers andM-MLV-Reverse Transcriptase (essentially as described by manufacturer(Ambion)) 0.25 μg total RNA of each sample was adjusted to 10.8 μl inH₂O. 2 μl decamers and 2 μl dNTP mix (2.5 mM each) was added. Sampleswere heated to 70° C. for 3 min. and cooled immediately in ice water andadded 3.25 μl of a mix containing (2 μl 10× RT buffer; 1 μl M-MLVReverse Transcriptase; 0.25 μl RNAase inhibitor). cDNA is synthesized at42° C. for 60 min followed by heating inactivation step at 95° C. for 10min and finally cooled to 4° C. The cDNA can further be used for mRNAquantification by for example Real-time quantitative PCR.

mRNA expression can be assayed in a variety of ways known in the art.For example, mRNA levels can be quantitated by, e.g., Northern blotanalysis, competitive polymerase chain reaction (PCR), Ribonucleaseprotection assay (RPA) or real-time PCR. Real-time quantitative PCR ispresently preferred. RNA analysis can be performed on total cellular RNAor mRNA.

Methods of RNA isolation and RNA analysis such as Northern blot analysisare routine in the art and is taught in, for example, Current Protocolsin Molecular Biology, John Wiley and Sons.

Real-time quantitative (PCR) can be conveniently accomplished using thecommercially available iQ Multi-Color Real Time PCR Detection Systemavailable from BioRAD. Real-time Quantitative PCR is a techniquewell-known in the art and is taught in for example Heid et al. Real timequantitative PCR, Genome Research (1996), 6: 986-994.

Example 11 LNA Oligonucleotide Uptake and Efficacy in vivo

In vivo study: Six groups of animals (5 mice per group) were treated inthe following manner. Group 1 animals were injected with 0.2 ml salineby i.v. on 3 successive days, Group 2 received 2.5 mg/kg SPC3372, Group3 received 6.25 mg/kg, Group 4 received 12.5 mg/kg and Group 5 received25 mg/kg, while Group 6 received 25 mg/kg SPC3373 (mismatch LNA-antimiR™oligonucleotide), all in the same manner. All doses were calculated fromthe Day 0 body weights of each animal.

Before dosing (Day 0) and 24 hour after last dose (Day 3), retro-orbitalblood was collected in tubes containing EDTA and the plasma fractionharvested and stored frozen −80° C. for cholesterol analysis. Atsacrifice livers were dissected and one portion was cut into 5 mm cubesand immersed in 5 volumes of ice-cold RNAlater. A second portion wassnap frozen in liquid nitrogen and stored for cryo-sectioning.

Total RNA was extracted from liver samples as described above andanalysed for miR-122a levels by microRNA specific QPCR. FIG. 5demonstrates a clear dose-response obtained with SPC3372 with an IC50 atca 3-5 mg/kg, whereas no miR-122a inhibition was detected using themismatch LNA antago-mir SPC3373 for miR-122a.

Example 12 LNA-antimiR-122a Dose-Response in vivo in C57/BL/J FemaleMice

In vivo study: Ten groups of animals (female C57/BL6; 3 mice per group)were treated in the following manner. Group 1 animals were injected with0.2ml saline by i.p. on day 0, day 2 and day 4. Groups 2-10 were dosedby i.p. with three different conc. (25 mg/kg, 5 mg/kg and 1 mg/kg) ofeither LNA antimiR-122a/SPC3372 (group 2-4), LNA antimir-122a/SPC3548(group 5-7) or LNA antimir-122a/SPC3549 (group 8-10); the LNAantimir-122a sequences are given in the Table 1. All three LNAantimiR-122a oligonucleotides target the liver-specific miR-122a. Thedoses were calculated from the Day 0 body weights of each animal.

The animals were sacrificed 48 hours after last dose (Day 6),retro-orbital blood was collected in tubes containing EDTA and theplasma fraction harvested and stored frozen −80° C. for cholesterolanalysis. At sacrifice livers were dissected and one portion was cutinto 5 mm cubes and immersed in 5 volumes of ice-cold RNAlater. A secondportion was snap frozen in liquid nitrogen and stored forcryo-sectioning.

Total RNA was extracted from liver samples using Trizol reagentaccording to the manufacturer's recommendations (Invitrogen, USA) andanalysed for miR-122a levels by microRNA-specific QPCR according to themanufacturer's recommendations (Ambion, USA). FIG. 2 demonstrates aclear dose-response obtained with all three LNA antimir-122a molecules(SPC3372, SPC3548, SPC3549). Both SPC3548 and SPC3549 show significantlyimproved efficacy in vivo in miR-122a silencing (as seen from thereduced miR-122a levels) compared to SPC3372, with SPC3549 being mostpotent (IC₅₀ ca 1 mg/kg).

The above example was repeated using SPC3372 and SPC3649 using 5 miceper group and the data combined (total of eight mice per group) is shownin FIG. 2 b.

Example 12a Northern Blot

MicroRNA specific northern blot showing enhanced miR-122 blocking bySPC3649 compared to SPC3372 in LNA-antimiR treated mouse livers.

Oligos used in this example:

SPC3649: 5′-CcAttGTcaCaCtCC-3′ New design (SEQ ID 59) SPC3372:5′-CcAttGtcAcaCtcCa-3′ Old design (SEQ ID 57)

We decided to assess the effect of SPC3649 on miR-122 miRNA levels inthe livers of SPC3649-treated mice. The LNA-antimiRs SPC3649 and SPC3372were administered into mice by three i.p. injections on every second dayover a six-day-period at indicated doses followed by sacrificing theanimals 48 hours after the last dose. Total RNA was extracted from thelivers. miR-122 levels were assessed by microRNA specific northern blot(FIG. 6)

Treatment of normal mice with SPC3649 resulted in dramatically improved,dose-dependent reduction of miR-122. MicroRNA specific northern blotcomparing SPC3649 with SPC3372 was performed (FIG. 6). SPC3649completely blocked miR-122 at both 5 and 25 mg/kg as seen by the absenceof mature single stranded miR-122 and only the presence of the duplexband between the LNA-antimiR and miR-122. Comparing duplex versus matureband on the northern blot SPC3649 seem equally efficient at 1 mg/kg asSPC3372 at 25 mg/kg.

Example 13 Assessment of Cholesterol Levels in Plasma in LNA Anti-miR122Treated Mice

Total cholesterol level was measured in plasma using a colometric assayCholesterol CP from ABX Pentra. Cholesterol was measured followingenzymatic hydrolysis and oxidation (2.3). 21.5 μl water was added to 1.5μl plasma. 250 μl reagent was added and within 5 min the cholesterolcontent measured at a wavelength of 540 nM. Measurements on each animalwere made in duplicate. The sensitivity and linearity was tested with2-fold diluted control compound (ABX Pentra N control). The cholesterollevel was determined by subtraction of the background and presentedrelative to the cholesterol levels in plasma of saline treated mice.

FIG. 3 demonstrates a markedly lowered level of plasma cholesterol inthe mice that received SPC3548 and SPC3549 compared to the salinecontrol at Day 6.

Example 14 Assessment of miR-122a Target mRNA Levels in LNA antimiR-122aTreated Mice

The saline control and different LNA-antimiR-122a treated animals weresacrificed 48 hours after last dose (Day 6), and total RNA was extractedfrom liver samples as using Trizol reagent according to themanufacturer's recommendations (Invitrogen, USA). The mRNA levels wereassessed by real-time quantitative RT-PCR for two miR-122a target genes,Bckdk (branched chain ketoacid dehydrogenase kinase, ENSMUSG00000030802)and aldolase A (aldoA, ENSMUSG00000030695), respectively, as well as forGAPDH as control, using Taqman assays according to the manufacturer'sinstructions (Applied biosystems, USA). FIGS. 4 a and 4 b demonstrate aclear dose-dependent upregulation of the two miR-122a target genes,Bckdk and AldoA, respectively, as a response to treatment with all threeLNA antimiR-122a molecules (SPC3372, SPC3548, SPC3549). In contrast, theqPCR assays for GAPDH control did not reveal any differences in the GAPDmRNA levels in the LNA-antimiR-122a treated mice compared to the salinecontrol animals (FIG. 4 c). The Bckdk and AldoA mRNA levels weresignificantly higher in the SPC3548 and SPC3549 treated mice compared tothe SPC3372 treated mice (FIGS. 4 a and 4 b), thereby demonstratingtheir improved in vivo efficacy.

Example 15 LNA Oligonucleotide Duration of Action in vivo

In vivo study: Two groups of animals (21 mice per group) were treated inthe following manner. Group 1 animals were injected with 0.2 ml salineby i.v. on 3 successive days, Group 2 received 25 mg/kg SPC3372 in thesame manner. All doses were calculated from the Day 0 body weights ofeach animal.

After last dose (Day 3), 7 animals from each group were sacrificed onDay 9, Day 16 and Day 23, respectively. Prior to this, on each day,retro-orbital blood was collected in tubes containing EDTA and theplasma fraction harvested and stored frozen −80° C. for cholesterolanalysis from each day. At sacrifice livers were dissected and oneportion was cut into 5 mm cubes and immersed in 5 volumes of ice-coldRNAlater. A second portion was snap frozen in liquid nitrogen and storedfor cryo-sectioning.

Total RNA was extracted from liver samples as described above andanalysed for miR-122a levels by microRNA specific QPCR. FIG. 7(Sacrifice day 9, 16 or 23 correspond to sacrifice 1, 2 or 3 weeks afterlast dose) demonstrates a two-fold inhibition in the mice that receivedSPC3372 compared to the saline control, and this inhibition could stillbe detected at Day 16, while by Day 23 the mi122a levels approachedthose of the saline group.

Example 16 LNA Oligonucleotide Duration of Action in vivo

In vivo study: Two groups of animals (21 mice per group) were treated inthe following manner. Group 1 animals were injected with 0.2 ml salineby i.v. on 3 successive days, Group 2 received 25 mg/kg SPC3372 in thesame manner. All doses were calculated from the Day 0 body weights ofeach animal.

After last dose (Day 3), 7 animals from each group were sacrificed onDay 9, Day 16 and Day 23, respectively. Prior to this, on each day,retro-orbital blood was collected in tubes containing EDTA and theplasma fraction harvested and stored frozen −80° C. for cholesterolanalysis from each day. At sacrifice livers were dissected and oneportion was cut into 5 mm cubes and immersed in 5 volumes of ice-coldRNAlater. A second portion was snap frozen in liquid nitrogen and storedfor cryo-sectioning.

Total RNA was extracted from liver samples as described above andanalysed for miR-122a levels by microRNA specific QPCR. FIG. 8demonstrates a two-fold inhibition in the mice that received SPC3372compared to the saline control, and this inhibition could still bedetected at Day 16, while by Day23 the miR-122a levels approached thoseof the saline group.

As to examples 17-22, the following procedures apply:

NMRI mice were administered intravenously with SPC3372 using daily dosesranging from 2.5 to 25 mg/kg for three consecutive days. Animals weresacrificed 24 hours, 1, 2 or 3 weeks after last dose. Livers wereharvested divided into pieces and submerged in RNAlater (Ambion) orsnap-frozen. RNA was extracted with Trizol reagent according to themanufacturer's instructions (Invitrogen) from the RNAlater tissue,except that the precipitated RNA was washed in 80% ethanol and notvortexed. The RNA was used for mRNA TaqMan qPCR according tomanufacturer (Applied biosystems) or northern blot (see below). Thesnap-frozen pieces were cryo-sectioned for in situ hybridizations.

Further, as to FIGS. 9-14, SPC3372 is designated LNA-antimiR and SPC3373(the mismatch control) is designated “mm” instead of using the SPCnumber.

Example 17 Dose Dependent miR-122a Target mRNA Induction by SPC3372Inhibition of miR-122a

Mice were treated with different SPC3372 doses for three consecutivedays, as described above and sacrificed 24 hours after last dose. TotalRNA extracted from liver was subjected to qPCR. Genes with predictedmiR-122 target site and observed to be upregulated by microarrayanalysis were investigated for dose-dependent induction by increasingSPC3372 doses using qPCR. Total liver RNA from 2 to 3 mice per groupsacrificed 24 hours after last dose were subjected to qPCR for theindicated genes. Shown in FIG. 9 is mRNA levels relative to Salinegroup, n=2-3 (2.5-12.5 mg/kg/day: n=2, no SD). Shown is also themismatch control (mm, SPC3373).

Assayed genes: Nrdg3 Aldo A, Bckdk, CD320 with predicted miR-122 targetsite. Aldo B and Gapdh do not have a predicted miR-122a target site.

A clear dose-dependent induction was seen of the miR-122a target genesafter treatment with different doses of SPC3372.

Example 18 Transient Induction of miR-122a Target mRNAs FollowingSPC3372 Treatment

NMRI female mice were treated with 25 mg/kg/day SPC3372 along withsaline control for three consecutive days and sacrificed 1, 2 or 3 weeksafter last dose, respectively. RNA was extracted from livers and mRNAlevels of predicted miR-122a target mRNAs, selected by microarray datawere investigated by qPCR. Three animals from each group were analysed.

Assayed genes: Nrdg3 Aldo A, Bckdk, CD320 with predicted miR-122 targetsite. Gapdh does not have a predicted miR-122a target site.

A transient induction followed by a restoration of normal expressionlevels in analogy with the restoration of normal miR-122a levels wasseen (FIG. 10).

mRNA levels are normalized to the individual GAPDH levels and to themean of the Saline treated group at each individual time point. Includedare also the values from the animals sacrificed 24 hours after lastdose. Shown is mean and standard deviation, n=3 (24 h n=3)

Example 19 Induction of Vldlr in Liver by SPC3372 Treatment

The same liver RNA samples as in previous example were investigated forVldlr induction.

A transient up-regulation was seen after SPC3372 treatment, as with theother predicted miR-122a target mRNAs (FIG. 11)

Example 20 Stability of miR-122a/SPC3372 Duplex in Mouse Plasma

Stability of SPC3372 and SPC3372/miR-122a duplex were tested in mouseplasma at 37° C. over 96 hours. Shown in FIG. 12 is a SYBR-Gold stainedPAGE.

SPC3372 was completely stable over 96 hours. The SPC3372/miR-122a duplexwas immediately truncated (degradation of the single stranded miR-122aregion not covered by SPC3372) but thereafter almost completely stableover 96 hours.

The fact that a preformed SPC3372/miR-122 duplex showed stability inserum over 96 hours together with the high thermal duplex stability ofSPC3372 molecule supported our notion that inhibition of miR-122a bySPC3372 was due to stable duplex formation between the two molecules,which has also been reported in cell culture (Naguibneva et al. 2006).

Example 21 Sequestering of Mature miR-122a by SPC3372 Leads to DuplexFormation

The liver RNA was also subjected to microRNA Northern blot. Shown inFIG. 13 is a membrane probed with a miR-122a specific probe (upperpanel) and re-probed with a Let-7 specific probe (lower panel). With themiR-122 probe, two bands could be detected, one corresponding to maturemiR-122 and one corresponding to a duplex between SPC3372 and miR-122.

To confirm silencing of miR-122, liver RNA samples were subjected tosmall RNA northern blot analysis, which showed significantly reducedlevels of detectable mature miR-122, in accordance with our real-timeRT-PCR results. By comparison, the levels of the let-7a control were notaltered. Interestingly, we observed dose-dependent accumulation of ashifted miR-122/SPC3372 heteroduplex band, suggesting that SPC3372 doesnot target miR-122 for degradation, but rather binds to the microRNA,thereby sterically hindering its function.

Northern blot analysis was performed as follows:

Preparation of northern membranes was done as described in Sempere etal. 2002, except for the following changes: Total RNA, 10 μg per lane,in formamide loading buffer (47.5% formamide, 9 mM EDTA, 0.0125%Bromophenol Blue, 0.0125% Xylene Cyanol, 0.0125% SDS) was loaded onto a15% denaturing Novex TBE-Urea polyacrylamide gel (Invitrogen) withoutpreheating the RNA. The RNA was electrophoretically transferred to aGeneScreen plus Hybridization Transfer Membrane (PerkinElmer) at 200 mAfor 35 min. Membranes were probed with 32P-labelled LNA-modifiedoligonucleotides complimentary to the mature microRNAs*. The LNAoligonucleotides were labelled and hybridized to the membrane asdescribed in (Válóczi et al. 2004) except for the following changes: Theprehybridization and hybridization solutions contained 50% formamide,0.5% SDS, 5×SSC, 5× Denhardt's solution and 20 μg/ml sheared denaturedherring sperm DNA. Hybridizations were performed at 45° C. The blotswere visualized by scanning in a Storm 860 scanner. The signal of thebackground membrane was subtracted from the radioactive signalsoriginating from the miRNA bands. The values of the miR-122 signals werecorrected for loading differences based on the let-7a signal. Todetermine the size of the radioactive signals the Decade Marker System(Ambion) was used according to the suppliers' recommendations.

Example 22 miR-122a Sequestering by SPC3372 Along with SPC3372Distribution Assessed by in Situ Hybridization of Liver Sections

Liver cryo-sections from treated animals were subjected to in situhybridizations for detection and localization of miR-122 and SPC3372(FIG. 14). A probe complementary to miR-122 could detect miR-122a. Asecond probe was complementary to SPC3372. Shown in FIG. 14 is anoverlay, in green is distribution and apparent amounts of miR-122a andSPC3372 and blue is DAPI nuclear stain, at 10x magnification. 100×magnifications reveal the intracellular distribution of miR-122a andSPC3372 inside the mouse liver cells.

The liver sections from saline control animals showed a strong miR-122staining pattern over the entire liver section, whereas the sectionsfrom SPC3372 treated mice showed a significantly reduced patchy stainingpattern. In contrast, SPC3372 molecule was readily detected in SPC3372treated liver, but not in the untreated saline control liver. Highermagnification localized miR-122a to the cytoplasm in the hepatocytes,where the miR-122 in situ pattern was clearly compartmentalized, whileSPC3372 molecule was evenly distributed in the entire cytoplasm.

Example 23 Micro Array Analysis

We carried out genome-wide expression profiling of total RNA samplesfrom saline LNA-antimiR-122 treated and LNA mismatch control treatedmice livers 24 hours after the last dose using Affymetrix Mouse Genome430 2.0 arrays. Analysis of the array data revealed 455 transcripts thatwere upregulated in the LNA-antimiR treated mice livers compared tosaline and LNA mismatch controls, while 54 transcripts weredownregulated (FIG. 15 a). A total of 415 of the upregulated and 53downregulated transcripts could be identified in the Ensembl database.We subsequently examined the 3′ untranslated regions (UTRs) of thedifferentially expressed mRNAs for the presence of the 6 nt sequenceCACTCC, corresponding to the reverse complement of the nucleotide 2-7seed region in mature miR-122. The number of transcripts having at leastone miR-122 recognition sequence was 213 (51%) among the upregulatedtranscripts, and 10 (19%) within the downregulated transcripts, whilethe frequency in a random sequence population was 25%, implying that asignificant pool of the upregulated mRNAs represent direct miR-122targets in the liver (FIG. 15 b).

The LNA-antimiR treatment showed maximal reduction of miR-122 levels at24 hours, 50% reduction at one week and matched saline controls at threeweeks after last LNA dose (Example 12 “old design”). This coincided witha markedly reduced number of differentially expressed genes between thetwo mice groups at the later time points. Compared to the 509 mRNAs 24hours after the last LNA dose we identified 251 differentially expressedgenes after one week, but only 18 genes after three weeks post treatment(FIGS. 15 c and 15 d). In general genes upregulated 24 hours afterLNA-antimiR treatment then reverted towards control levels over the nexttwo weeks (FIG. 15 d).

In conclusion, a large portion of up-regulated/de-repressed genes afterLNA-antimiR treatment are miR-122 targets, indicating a very specificeffect for blocking miR-122. Also genes up-regulated/de-repressedapproach normal levels 3 weeks after end of treatment, suggest arelative long therapeutic effect, but however not cause a permanentalteration, ie the effect is reversible.

Methods:

Gene expression profiling of LNA-antimiR treated mice.

Expression profiles of livers of saline and LNA-antimiR treated micewere compared. NMRI female mice were treated with 25 mg/kg/day ofLNA-antimiR along with saline control for three consecutive days andsacrificed 24 h, 1, 2 or 3 weeks after last dose. Additionally,expression profiles of livers of mice treated with the mismatch LNAcontrol oligonucleotide 24 h after last dose were obtained. Three micefrom each group were analyzed, yielding a total of 21 expressionprofiles. RNA quality and concentration was measured using an Agilent2100 Bioanalyzer and Nanodrop ND-1000, respectively. Total RNA wasprocessed following the GeneChip Expression 3′-Amplification ReagentsOne-cycle cDNA synthesis kit instructions (Affymetrix Inc, Santa Clara,Calif., USA) to produce double-stranded cDNA. This was used as atemplate to generate biotin-labeled cRNA following manufacturer'sspecifications. Fifteen micrograms of biotin-labeled cRNA was fragmentedto strands between 35 and 200 bases in length, of which 10 microgramswere hybridised onto Affymetrix Mouse Genome 430 2.0 arrays overnight inthe GeneChip Hybridisation oven 6400 using standard procedures. Thearrays were washed and stained in a GeneChip Fluidics Station 450.Scanning was carried out using the GeneChip Scanner 3000 and imageanalysis was performed using GeneChip Operating Software. Normalizationand statistical analysis were done using the LIMMA software package forthe R programming environment27. Probes reported as absent by GCOSsoftware in all hybridizations were removed from the dataset.Additionally, an intensity filter was applied to the dataset to removeprobes displaying background-corrected intensities below 16. Data werenormalized using quantile normalization28. Differential expression wasassessed using a linear model method. P values were adjusted formultiple testing using the Benjamini and Hochberg. Tests were consideredto be significant if the adjusted p values were p<0.05. Clustering andvisualization of Affymetrix array data were done using theMultiExperiment Viewer software29.

Target Site Prediction

Transcripts with annotated 3′ UTRs were extracted from the Ensembldatabase (Release 41) using the EnsMart data mining tool30 and searchedfor the presence of the CACTCC sequence which is the reverse complementof the nucleotide 2-7 seed in the mature miR-122 sequence. As abackground control, a set of 1000 sequences with a length of 1200 nt,corresponding to the mean 3′ UTR length of the up- and downregulatedtranscripts at 24 h after last LNA-antimiR dose, were searched for the 6nucleotide miR-122 seed matches. This was carried out 500 times and themean count was used for comparison

Example 24 Dose-Dependent Inhibition of miR-122 in Mouse Liver byLNA-antimiR is Enhanced as Compared to Antagomir Inhibition of miR-122

NMRI female mice were treated with indicated doses of LNA-antimiR(SPC3372) along with a mismatch control (mm, SPC3373), saline andantagomir (SPC3595) for three consecutive days and sacrificed 24 hoursafter last dose (as in example 11 “old design”, n=5). miR-122 levelswere analyzed by qPCR and normalized to the saline treated group. Geneswith predicted miR-122 target site and up regulated in the expressionprofiling (AldoA, Nrdg3, Bckdk and CD320) showed dose-dependentde-repression by increasing LNA-antimiR doses measured by qPCR.

The de-repression was consistently higher on all tested miR-122 targetmRNAs (AldoA, Bckdk, CD320 and Nrdg3 FIG. 17, 18, 19, 20) in LNA-antimiRtreated mice compared to antagomir treated mice. This was also indicatedwhen analysing the inhibition of miR-122 by miR-122 specific qPCR (FIG.16). Hence LNA-antimiRs give a more potent functional inhibition ofmiR-122 than corresponding dose antagomir.

Example 25 Inhibition of miR-122 by LNA-antimiR in HypercholesterolemicMice Along with Cholesterol Reduction and miR-122 Target mRNADe-Repression

C57BL/6J female mice were fed on high fat diet for 13 weeks before theinitiation of the SPC3649 treatment. This resulted in increased weightto 30-35 g compared to the weight of normal mice, which was just under20 g, as weighed at the start of the LNA-antimiR treatment. The high fatdiet mice lead to significantly increased total plasma cholesterol levelof about 130 mg/dl, thus rendering the mice hypercholesterolemiccompared to the normal level of about 70 mg/dl. Bothhypercholesterolemic and normal mice were treated i.p. twice weekly with5 mg/kg SPC3649 and the corresponding mismatch control SPC3744 for astudy period of 5½ weeks. Blood samples were collected weekly and totalplasma cholesterol was measured during the entire course of the study.Upon sacrificing the mice, liver and blood samples were prepared fortotal RNA extraction, miRNA and mRNA quantification, assessment of theserum transaminase levels, and liver histology.

Treatment of hypercholesterolemic mice with SPC3649 resulted inreduction of total plasma cholesterol of about 30% compared to salinecontrol mice already after 10 days and sustained at this level duringthe entire study (FIG. 21). The effect was not as pronounced in thenormal diet mice. By contrast, the mismatch control SPC3744 did notaffect the plasma cholesterol levels in neither hypercholesterolemic nornormal mice.

Quantification of miR-122 inhibition and miR-122 target gene mRNAde-repression (AldoA and Bckdk) after the long-term treatment withSPC3649 revealed a comparable profile in both hypercholesterolemic andnormal mice (FIG. 22, 23, 24), thereby demonstrating the potency ofSPC3649 in miR-122 antagonism in both animal groups. The miR-122 qPCRassay indicated that also the mismatch control SPC3744 had an effect onmiR-122 levels in the treated mice livers, albeit to a lesser extentcompared to SPC3649. This might be a reduction associated with thestem-loop qPCR. Consistent with this notion, treatment of mice with themismatch control SPC3744 did not result in any functional de-repressionof the direct miR-122 target genes (FIGS. 23 and 24) nor reduction ofplasma cholesterol (FIG. 21), implying that SPC3649-mediated antagonismof miR-122 is highly specific in vivo.

Liver enzymes in hypercholesterolemic and normal mice livers wereassessed after long term SPC3649 treatment. No changes in the alanineand aspartate aminotransferase (ALT and AST) levels were detected in theSPC3649 treated hypercholesterolemic mice compared to saline controlmice (FIGS. 25 and 26). A possibly elevated ALT level was observed inthe normal mice after long-term treatment with SPC3649 (FIG. 26).

Example 26 Methods for Performing the LNA-antimiR/HypercholesterolemicExperiment and Analysis

Mice and Dosing.

C57BL/6J female mice (Taconic M&B Laboratory Animals, Ejby, Denmark)were used. All substances were formulated in physiological saline (0.9%NaCl) to final concentration allowing the mice to receive anintraperitoneal injection volume of 10 ml/kg.

In the diet induced obesity study, the mice received a high fat (60EN %)diet (D12492, Research Diets) for 13 weeks to increase their bloodcholesterol level before the dosing started. The dose regimen wasstretched out to 5½ weeks of 5 mg/kg LNA-antimiR™ twice weekly. Bloodplasma was collected once a week during the entire dosing period. Aftercompletion of the experiment the mice were sacrificed and RNA extractedfrom the livers for further analysis. Serum was also collected foranalysis of liver enzymes.

Total RNA Extraction.

The dissected livers from sacrificed mice were immediately stored in RNAlater (Ambion). Total RNA was extracted with Trizol reagent according tothe manufacturer's instructions (Invitrogen), except that theprecipitated RNA pellet was washed in 80% ethanol and not vortexed.

MicroRNA-Specific Quantitative RT-PCR.

The miR-122 and let-7a microRNA levels were quantified with TaqManmicroRNA Assay (Applied Biosystems) following the manufacturer'sinstructions. The RT reaction was diluted ten times in water andsubsequently used for real time PCR amplification according to themanufacturer's instructions. A two-fold cDNA dilution series from livertotal RNA of a saline-treated animal or mock transfected cells cDNAreaction (using 2.5 times more total RNA than in samples) served asstandard to ensure a linear range (Ct versus relative copy number) ofthe amplification. Applied Biosystems 7500 or 7900 real-time PCRinstrument was used for amplification.

Quantitative RT-PCR

mRNA quantification of selected genes was done using standard TaqManassays (Applied Biosystems). The reverse transcription reaction wascarried out with random decamers, 0.5 pg total RNA, and the M-MLV RTenzyme from Ambion according to a standard protocol. First strand cDNAwas subsequently diluted 10 times in nuclease-free water before additionto the RT-PCR reaction mixture. A two-fold cDNA dilution series fromliver total RNA of a saline-treated animal or mock transfected cellscDNA reaction (using 2.5 times more total RNA than in samples) served asstandard to ensure a linear range (Ct versus relative copy number) ofthe amplification. Applied Biosystems 7500 or 7900 real-time PCRinstrument was used for amplification.

Metabolic Measurements.

Immediately before sacrifice retro-orbital sinus blood was collected inEDTA-coated tubes followed by isolation of the plasma fraction. Totalplasma cholesterol was analysed using ABX Pentra Cholesterol CP (HoribaGroup, Horiba ABX Diagnostics) according to the manufacturer'sinstructions.

Liver Enzymes (ALT and AST) Measurement

Serum from each individual mouse was prepared as follows: Blood sampleswere stored at room temperature for 2 h before centrifugation (10 min,3000 rpm at room temperature). After centrifugation, serum was harvestedand frozen at −20° C.

ALT and AST measurement was performed in 96-well plates using ALT andAST reagents from ABX Pentra according to the manufacturer'sinstructions. In short, serum samples were diluted 2.5 fold with H₂O andeach sample was assayed in duplicate. After addition of 50 μl dilutedsample or standard (multical from ABX Pentra) to each well, 200 μl of37° C. AST or ALT reagent mix was added to each well. Kineticmeasurements were performed for 5 min with an interval of 30 s at 340 nmand 37° C. using a spectrophotometer.

Example 27

Modulation of Hepatitis C Replication by LNA-antimiR (SPC3649)

Oligos used in this example (uppercase: LNA, lowercase DNA, LNA Cs aremethyl, and LNAs are preferably B-D-oxy (o subscript after LNA residue):

SPC3649 (LNA-antimiR targeting miR-122, was in the initial small scalesynthesis designated SPC3549) 5′-

c_(s)

t_(s)t_(s)

c_(s)a_(s)

a_(s)

t_(s)

-3′ SPC3648 (LNA-antimiR targeting miR-122, was in the initial smallscale synthesis designated SPC3548) 5′-

t_(s) t_(s)

c_(s) a_(s)

a_(s)

t_(s)

-3′ SPC3550 (4 nt mismatch control to SPC3649) SEQ ID 63 5′-

c_(s)

t_(s) t_(s)

g_(s) a_(s)

c_(s)

t_(s)

-3′ 2′OMe anti-122: full length (23 nt) 2′OMe modified oligocomplementary to miR-122 2′OMe Ctrl: scrambled 2′OMe modified control

Hepatitis C (HCV) replication has been shown to be facilitated bymiR-122 and consequently, antagonizing miR-122 has been demonstrated toaffect HCV replication in a hepatoma cell model in vitro. We assess theefficacy of SPC3649 reducing HCV replication in the Huh-7 based cellmodel. The different LNA-antimiR molecules along with a 2′OMe antisenseand scramble oligonucleotide are transfected into Huh-7 cells, HCV isallowed to replicate for 48 hours. Total RNA samples extracted from theHuh-7 cells are subjected to Northern blot analysis.

Example 28 Enhanced LNA-antimiR™ Antisense Oligonucleotide TargetingmiR-21

Mature miR-21 Sequence from Sanger Institute miRBase:

>hsa-miR-21 MIMAT0000076 UAGCUUAUCAGACUGAUGUUGA (SEQ ID NO4) >mmu-miR-21 MIMAT0000530 UAGCUUAUCAGACUGAUGUUGA (SEQ ID NO 64)

Sequence of Compounds:

SPC3521 miR-21 5′-FAM TCAgtctgataaGCTa-3′ (gap-mer design) - (SEQ ID NO65) SPC3870 miR-21(mm) 5′-FAM TCCgtcttagaaGATa-3′ - (SEQ ID NO 66)SPC3825 miR-21 5′-FAM TcTgtCAgaTaCgAT-3′ (new design) (SEQ ID NO 67)SPC3826 miR-21(mm) 5′-FAM TcAgtCTgaTaAgCT-3′- (SEQ ID NO 68) SPC3827miR-21 5′-FAM TcAGtCTGaTaAgCT-3′ (new, enhanced design) - (SEQ ID NO 69)

All compounds have a fully or almost fully thiolated backbone and havehere also a FAM label in the 5′ end.

miR-21 has been show to be up-regulated in both glioblastoma (Chan etal. Cancer Research 2005, 65 (14), p6029) and breast cancer (Iorio etal. Cancer Research 2005, 65 (16), p7065) and hence has been considereda potential ‘oncogenic’ microRNA. Chan et al. also show induction ofapoptosis in glioblastoma cells by antagonising miR-21 with 2′OMe or LNAmodified antisense oligonucleotides. Hence, agents antagonising miR-21have the potential to become therapeutics for treatment of glioblastomaand other solid tumours, such as breast cancer. We present an enhancedLNA modified oligonucleotide targeting miR-21, an LNA-antimiR™, withsurprisingly good properties to inhibit miR-21 suited for theabovementioned therapeutic purposes.

Suitable therapeutic administration routes are, for example,intracranial injections in glioblastomas, intratumoural injections inglioblastoma and breast cancer, as well as systemic delivery in breastcancer

Inhibition of miR-21 in 0373 Glioblastoma Cell Line and MCF-7 BreastCancer Cell Line

Efficacy of current LNA-anitmiR™ is assessed by transfection atdifferent concentrations, along with control oligonucleotides, into U373and MCF-7 cell lines known to express miR-21 (or others miR-21expressing cell lines as well). Transfection is performed using standardLipofectamine2000 protocol (Invitrogen). 24 hours post transfection, thecells are harvested and total RNA extracted using the Trizol protocol(Invitrogen). Assessment of miR-21 levels, depending on treatment andconcentration used is done by miR-21 specific, stem-loop real-timeRT-PCR (Applied Biosystems), or alternatively by miR-21 specificnon-radioactive northern blot analyses. The detected miR-21 levelscompared to vehicle control reflects the inhibitory potential of theLNA-antimiR™.

Functional Inhibition of miR-21 by Assessment of miR-21 Target GeneUp-Regulation.

The effect of miR-21 antagonism is investigated through cloning of theperfect match miR-21 target sequence behind a standard Renillaluciferase reporter system (between coding sequence and 3′ UTR,psiCHECK-2, Promega)—see Example 29. The reporter construct andLNA-antimiR™ will be co-transfected into miR-21 expressing cell lines(f. ex. U373, MCF-7). The cells are harvested 24 hours post transfectionin passive lysis buffer and the luciferase activity is measuredaccording to a standard protocol (Promega, Dual Luciferase ReporterAssay System). The induction of luciferase activity is used todemonstrate the functional effect of LNA-antimiR™ antagonising miR-21.

Example 29 Luciferase Reporter Assay for Assessing Functional Inhibitionof MicroRNA by LNA-antimiRs and Other MicroRNA Targeting Oligos:Generalisation of New and Enhanced New Design as Preferred Design forBlocking MicroRNA Function

Oligos used in this example (uppercase: LNA, lowercase: DNA) to assessLNA-antimiR de-repressing effect on luciferase reporter with microRNAtarget sequence cloned by blocking respective microRNA:

Oligo #, target microRNA, oligo sequence Design target: hsa-miR-122aMIMAT0000421 uggagugugacaaugguguuugu screened in HUH-7 cell lineexpressing miR-122 3962: miR-122 5′-ACAAacaccattgtcacacTCCA-3′ Fullcomplement, gap 3965: miR-122 5′-acaaacACCATTGTcacactcca- Fullcomplement, block 3′ 3972: miR-122 5′-acAaaCacCatTgtCacActCca-3′ Fullcomplement, LNA_3 3549 (3649): miR-122 5′-CcAttGTcaCaCtCC-3′ New design3975: miR-122 5′-CcAtTGTcaCACtCC-3′ Enhanced new design target:hsa-miR-19b MIMAT0000074 ugugcaaauccaugcaaaacuga screened HeLa cell lineexpressing miR-19b 3963: miR-19b 5′-TCAGttttgcatggatttgCACA-3′ Fullcomplement, gap 3967: miR-19b 5′-tcagttTTGCATGGatttgcaca-3′ Fullcomplement, block 3973: miR-19b 5′-tcAgtTttGcaTggAttTgcAca-3′ Fullcomplement, LNA_3 3560: miR-19b 5′-TgCatGGatTtGcAC-3′ New design 3976:miR-19b 5′- Enhanced new design TgCaTGGatTTGcAC-3′ target: hsa-miR-155MIMAT0000646 uuaaugcuaaucgugauagggg screen in 518A2 cell line expressingmiR-155 3964: miR-155 5′-CCCCtatcacgattagcaTTAA-3′ Full complement, gap3968: miR-155 5′-cccctaTCACGATTagcattaa-3′ Full complement, block 3974:miR-155 5′-cCccTatCacGatTagCatTaa-3′ Full complement, LNA_3 3758:miR-155 5′-TcAcgATtaGcAtTA-3′ New design 3818: miR-1555′-TcAcGATtaGCAtTA-3′ Enhanced new design SEQ ID NOs as before.

A reporter plasmid (psiCheck-2 Promega) encoding both the Renilla andthe Firefly variants of luciferase was engineered so that the 3′UTR ofthe Renilla luciferase includes a single copy of a sequence fullycomplementary to the miRNA under investigation.

Cells endogenously expressing the investigated miRNAs (HuH-7 formiR-122a, HeLa for miR-19b, 518A2 for miR-155) were co-transfected withLNA-antimiRs or other miR binding oligonucleotides (the fullcomplementary ie full length) and the corresponding microRNA targetreporter plasmid using Lipofectamine 2000 (Invitrogen). The transfectionand measurement of luciferase activity were carried out according to themanufacturer's instructions (Invitrogen Lipofectamine 2000/PromegaDual-luciferase kit) using 150 000 to 300 000 cells per well in 6-wellplates. To compensate for varying cell densities and transfectionefficiencies the Renilla luciferase signal was normalized with theFirefly luciferase signal. All experiments were done in triplicate.

Surprisingly, new design and new enhanced design were the bestfunctional inhibitors for all three microRNA targets, miR-155, miR-19band miR-122 (FIG. 27, 28, 29). The results are summarized in followingtable 3.

Result Summary:

TABLE 3 Degree of de-repression of endogenous miR-155, miR-19b andmiR-122a function by various designs of LNA-antimiR's. Design miR-155miR-19b miR-122a New enhanced design *** *** no data New design *** ****** Full complement, LNA_3 ** *** ** Full complement, block ** ** **Full complement, gap * not signif. not signif.

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1-133. (canceled)
 134. An oligonucleotide with a length of 8-26nucleobase units, wherein the oligonucleotide comprises a corenucleobase sequence from positions two to seven or from positions threeto eight, counting from 3′ end of the oligonucleotide, selected from thegroup consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9, and SEQ ID NO: 10, wherein at least two nucleobase units in said coresequence are nucleotide analogues, and wherein said oligonucleotidecomprises at least one non-phosphodiester internucleoside linkage. 135.The oligonucleotide of claim 134, comprising a nucleobase sequenceselected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, SEQID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 28,SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO:84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ IDNO: 89, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19,SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO:94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ IDNO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 29, SEQ ID NO: 30,SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO:35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 102, SEQ ID NO: 103, SEQ IDNO: 104, SEQ ID NO: 105, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49,SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO:54, SEQ ID NO: 55, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQID NO: 109, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68,SEQ ID NO: 69, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO:41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQID NO:
 46. 136. The oligonucleotide of claim 134, wherein at least oneinternucleoside linkage is selected from the group consisting of(—O—P(O)₂—O—)—, —O—P(O,S)—O—, —O—P(S)₂—O—, —S—P(O)₂—O—, —S—P(O,S)—O—,—S—P(S)₂—O—, —O—P(O)₂—S—, —O—P(O,S)—S—, —S—P(O)₂—S—, —O—PO(R^(H))—O—,O—PO(OCH₃)—O—, —O—PO(NR^(H))—O—, —O—PO(OCH₂CH₂S—R)—O—, —O—PO(BH₃)—O—,—O—PO(NHR^(H))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—, —NR^(H)—CO—O—,—NR^(H)—CO—NR^(H)—, —O—CO—O—, —O—CO—NR^(H)—, —NR^(H)—CO—CH₂—,—O—CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—, —CO—NR^(H)—CH₂—, —CH₂—NR^(H)—CO—,—O—CH₂—CH₂—S—, —S—CH₂—CH₂—O—, —S—CH₂—CH₂—S—, —CH₂—SO₂—CH₂—,—CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—CO—, —CH₂—NCH₃—O—CH₂—, wherein is Hor C_(1-4-alkyl), and combinations thereof.
 137. The oligonucleotide ofclaim 136, wherein at least one internucleoside linkage is —O—P(O,S)—O—(phosphorothioate).
 138. The oligonucleotide of claim 137, wherein theoligonucleotide is fully phosphorothioated.
 139. The oligonucleotide ofclaim 134, wherein each said at least two nucleotide analogues in saidcore nucleobase sequence is independently selected from the groupconsisting of a 2′-O-alkyl-RNA unit, a 2′-amino-DNA unit, a2′-fluoro-DNA unit, an LNA unit, a PNA unit, an HNA unit, and an INAunit.
 140. The oligonucleotide of claim 139, wherein each of said atleast two nucleotide analogues in said core nucleobase sequence is anLNA unit.
 141. The oligonucleotide of claim 140, wherein the at leasttwo LNA units in the core sequence are separated by at least one DNAunit.
 142. The oligonucleotide of claim 134, wherein the nucleobaseunits of said oligonucleotide that are not nucleotide analogues are DNAunits.
 143. The oligonucleotide of claim 142, wherein no more than 4consecutive nucleobase units in said oligonucleotide are DNA units. 144.The oligonucleotide of claim 134, wherein the first nucleobase unit,counting from 3′-end of the oligonucleotide, is an LNA unit.
 145. Theoligonucleotide of claim 134, wherein the first nucleobase unit,counting from 5′-end of the oligonucleotide, is an LNA unit.
 146. Theoligonucleotide of claim 134, wherein a nucleobase unit at positionnine, counting from 3′-end of the oligonucleotide, is an LNA unit. 147.The oligonucleotide of claim 146, wherein a nucleobase unit at positionten, counting from 3′-end of the oligonucleotide, is an LNA unit. 148.The oligonucleotide of claim 134, comprising the core nucleobasesequence from positions two to seven.
 149. The oligonucleotide of claim134, wherein the oligonucleotide does not mediate RNase H cleavage of acomplementary single stranded RNA molecule.
 150. The oligonucleotide ofclaim 134, wherein no more than two consecutive nucleobase units are LNAunits.
 151. The oligonucleotide of claim 134, further comprising anon-nucleotide or non-polynucleotide moiety conjugated to saidoligonucleotide.
 152. The oligonucleotide of claim 134, with a length of14 to 16 nucleobase units.
 153. The oligonucleotide of claim 134,wherein the sequence from positions two or three, counting from 3′ endof the oligonucleotide, is selected from the group consisting of SEQ IDNO: 73, SEQ ID NO: 74, and SEQ ID NO:
 75. 154. A composition comprisingthe oligonucleotide according to claim 134, and pharmaceuticallyacceptable diluents, carriers or adjuvants.
 155. A method of treating adisease or a disorder associated with a presence or an increaseexpression of miR-122, said method comprising administering to a personin need thereof an effective amount of the composition of claim 154.156. The method of claim 155, wherein the disease is a metabolicdisorder.
 157. The method of claim 155, wherein the disease is ahypercholesterolemia.
 158. The method of claim 155, wherein the diseaseis hepatitis C.
 159. A method of reducing microRNA-122 activity in acell which is expressing microRNA-122, comprising contacting the cellwith an effective amount of the composition of claim
 154. 160. A methodfor making the oligonucleotide of claim 134, said method comprising thesteps of: a. Selecting a region of the oligonucleotide which correspondsto any one of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, orSEQ ID NO: 10; b. Selecting one or more nucleobase units selected fromthe group consisting of 2′-O-alkyl-RNA unit, a 2′-amino-DNA unit, a2′-fluoro-DNA unit, an LNA unit, a PNA unit, an HNA unit, an INA unit,and combinations thereof, in positions 3 to 8, counting from the 3′-end;and c. synthesizing the regions defined in steps a and b.
 161. Anoligonucleotide of the formula: 5′-CcAttGTcaCaCtCC-3′, (SEQ ID NO: 99)

wherein: a lowercase letter identifies a nitrogenous base of a DNA unit;an uppercase bold letter identifies the nitrogenous base of an LNA unit;and one or more of LNA cytosines are optionally methylated.
 162. Theoligonucleotide according to claim 161, comprising at least onephosphorothioate internucleoside linkage.
 163. The oligonucleotideaccording to claim 161, comprising phosphodiester and phosphorothioateinternucleoside linkages.
 164. The oligonucleotide according to claim162, wherein all the internucleoside linkages are phosphorothioatelinkages.
 165. The oligonucleotide according to claim 164, wherein theLNA cytosines are 5-methylcytosines.
 166. The oligonucleotide accordingto claim 164, wherein the LNA units are beta-D-oxy LNA units.
 167. Theoligonucleotide according to claim 165, wherein the LNA units arebeta-D-oxy LNA units.
 168. The oligonucleotide according to claim 161 ofthe formula: (SEQ ID NO: 59) 5′-

c_(s)

t_(s)t_(s)

c_(s)a_(s)

a_(s)

t_(s)

-3′

wherein: a lowercase letter identifies a DNA unit; an upper case boldletter identifies an LNA unit; ^(m)C identifies a 5-methylcytosine LNA;subscript_(s) identifies a phosphorothioate internucleoside linkage, andthe LNA units are beta-D-oxy, as identified by a ⁰superscript after theLNA units.
 169. A composition comprising the oligonucleotide accordingto claim 161, and a pharmaceutically acceptable diluent, carrier oradjuvant, or combinations thereof.
 170. A composition comprising theoligonucleotide according to claim 168, and a pharmaceuticallyacceptable diluent, carrier or adjuvant, or combinations thereof.
 171. Amethod of treating a disease or a disorder associated with a presence oran increase expression of miR-122, comprising administering to a personin need thereof an effective amount of the composition of claim 169.172. A method of treating a disease or a disorder associated with apresence or an increase expression of miR-122, comprising administeringto a person in need thereof an effective amount of the composition ofclaim
 170. 173. The method of claim 172, wherein the disease is ametabolic disorder.
 174. The method of claim 172, wherein the disease isa hypercholesterolemia.
 175. The method of claim 172, wherein thedisease is hepatitis C infection.
 176. A method of reducing microRNA-122activity in a cell which is expressing microRNA-122, comprisingcontacting the cell with an effective amount of the composition of claim169.